[Cellular interactions in the vascular wall and mechanisms of action of endothelial factors].
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Animals↗
Biomedical subjects
U Walter
Publications and source records attributed to U Walter.
[Development of a health promotion program for the target group "elderly people"].
The importance of health promotion for persons in the second half of their life span is increasing. Results of different studies published at the end of the eighties show that health risks and anatomical deterioration can be effectively reduced in later life. From a medical viewpoint, these results provide the basis for preventive and rehabilitative measures. Knowledge from gerontology, social sciences and psychology demonstrates the necesserity of health promotion including the total way of life. First, the demographic and social framework for a concept of health promotion will be presented. The article will start with a short overview of the demographic-historical situation and the focus on the situations of different generations. Finally, the concrete requirements for the development of health promotion programmes will be formulated.
Aged↗
Synergistic phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein in intact human platelets in response to cGMP- and cAMP-elevating platelet inhibitors.
The mechanism underlying the synergistic inhibition of platelet activation by cGMP- and cAMP-elevating vasodilators was investigated using washed human platelets and platelet-rich plasma. With both types of human platelet preparations, low concentrations of sodium nitroprusside increased the cAMP-elevating potency of low concentrations of prostaglandin E1 (PG-E1). Using threshold concentrations of both sodium nitroprusside and PG-E1, the NO-donor potentiated the effect of PG-E1 with respect to the phosphorylation of the focal adhesion-associated vasodilator-stimulated phosphoprotein (VASP) at serine157. In contrast, threshold concentrations of cell-membrane permeant selective activators of the platelet cGMP-dependent protein kinase or the cAMP-dependent protein kinase had only additive effects on VASP serine157 phosphorylation in washed human platelets. The data demonstrate that low intracellular levels of cGMP effectively inhibit type III cGMP-inhibited phosphodiesterase in human platelets despite the high levels of cGMP-dependent protein kinase present in this cell type. This study provides the first evidence that the simultaneous activation of both cGMP- and cAMP-dependent protein kinase results in additive effects on VASP serine157 phosphorylation, whereas the supra-additive effects observed with the combination of sodium nitroprusside and PG-E1 are due to cGMP-mediated inhibition of type III phosphodiesterase. VASP phosphorylation at serine157 may be an important component underlying the synergistic inhibition of human platelets by cGMP-and cAMP-elevating agents.
Alprostadil↗
Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition.
Integrins and other adhesion receptors are essential components for outside-in and inside-out signaling through the cell membrane. The platelet glycoprotein IIb-IIIa (also known as fibrinogen receptor or integrin alpha IIb beta 3) is activated by platelet agonists, inhibited by cyclic-nucleotide-elevating agents, and is involved in the activation of protein tyrosine kinases including the 125-kDa focal adhesion kinase (pp125FAK). However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets cAMP- and cGMP-dependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-filtered platelets, cAMP-elevating agents [e.g. prostaglandin E1 and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to 70-100%. NO-generating, cGMP-elevating agents [e.g. 3-morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to a maximal extent of 30-50%. The effects of cAMP- and cGMP-elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane-permeant selective activators of platelet cAMP- or cGMP-dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb-IIIa inhibition. It is proposed that the inhibition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves cAMP-and cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.
Adenosine Diphosphate↗
Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II.
The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse transcriptase PCR and oligonucleotide primers encoding two conserved motifs of known cGMP-dependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this clone, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKII isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat motif that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK II localization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK II poly(A) RNA form was 4.8 kb, with minor forms of 6.2 and 3.1 kb. The cGK II RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.
Amino Acid Sequence↗
Indirect regulation of Ca2+ entry by cAMP-dependent and cGMP-dependent protein kinases and phospholipase C in rat platelets.
The Ca2+ responses of rat platelets are dominated by the influx of extracellular Ca2+ across the plasma membrane [Heemskerk, J. W. M., Feijge, M. A. H., Rietman, E. & Hornstra, G. (1991) FEBS Lett. 284, 223], which allows the study of Ca2+ entry into these cells by measuring increases in cytosolic Ca2+ concentration, [Ca2+]i. Several pieces of evidence indicated that, as in human platelets [Sage, S. O., Reast, R., & Rink, T. J. (1990) Biochem. J. 265, 675-680; Alonso, M., Alvarez, J., Montero, M., Sanchez, A. & García-Sancho, J. (1991) Biochem. J. 280, 783-789], agonist-stimulated Ca2+ entry was linked to the mobilisation of Ca2+ from intracellular stores: there was good correlation between the potency of receptor agonists in elevating [Ca2+]i in the presence or absence of external CaCl2; agonist-induced Ca2+ entry was inhibited to a similar degree as internal mobilisation by activators of cAMP-dependent or cGMP-dependent protein kinase or by the phospholipase C inhibitor, U73122; thapsigargin (an inhibitor of endomembrane Ca(2+)-ATPases) evoked store depletion and Ca2+ entry, which were both reduced by prior activation of cAMP-dependent or cGMP-dependent protein kinase but were not affected by U73122. In platelets with depleted Ca2+ stores, the addition of CaCl2 resulted in a considerable entry of Ca2+ which was insensitive to cAMP-dependent and cGMP-dependent protein kinase activation. In control platelets with full Ca2+ stores, CaCl2 potentiated the thrombin-induced generation of myo-inositol phosphates, suggesting that Ca2+ entry potentiated phospholipase C activity. Taken together, these results indicate that Ca2+ entry in rat platelets, (a) is mostly secondary to store depletion, (b) is not directly downregulated by cAMP-dependent and cGMP-dependent protein kinase, but indirectly by inhibition of store depletion, (c) can proceed in the absence of phospholipase C activation, but is stimulated by this activity probably by increased mobilisation of Ca2+ from the stores. These results lead to the concept that a major part of receptor-mediated Ca2+ entry in rat platelets is regulated in an indirect way by factors that stimulate or inhibit the degree of Ca2+ mobilisation from the internal stores.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets.
The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cAMP-dependent- (cAK) and cGMP-dependent protein kinase (cGK) in human platelets and other cardiovascular cells. To identify the VASP phosphorylation sites, purified VASP was phosphorylated by either protein kinase and subjected to trypsin, V8 and Lys-C proteolysis. The phosphorylated proteolytic fragments obtained were separated by reversed phase high performance liquid chromatography. Sequence analysis of the phosphorylated peptides and 32P measurement of the released 32P-labeled amino acids revealed three phosphorylation sites: a serine 1-containing site (LRKVSKQEEA), a serine 2-containing site (HIERRVSNAG), and a threonine-containing site (MNAVLARRRKATQVGE). Additional experiments with purified VASP demonstrated that both cAK and cGK phosphorylated serine 2 rapidly and the threonine residue slowly, whereas cGK phosphorylated the serine 1 residue more rapidly than the cAK. These differences in the phosphorylation rates of VASP by the two protein kinases were also observed with synthetic peptides corresponding to the sequences of the three identified phosphorylation sites. These experiments also established the synthetic peptide serine 1 as one of the best in vitro cGK substrates and the serine 2-containing site as the site responsible for the phosphorylation-induced mobility shift of VASP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with 32P-labeled platelets provided evidence that VASP is phosphorylated at the same three identified sites also in intact cells and that selective activation of cAK or cGK primarily increased the phosphorylation of both serine 2 and serine 1 but not threonine. Our results demonstrated overlapping substrate specificities of cAK and cGK in vitro and in intact cells. However, important quantitative and qualitative differences between cAK- and cGK-mediated phosphorylation of the focal adhesion protein VASP in human platelets were also observed, suggesting distinct functions of the two types of cyclic nucleotide-mediated VASP phosphorylation.
Amino Acid Sequence↗
Valence of praseodymium in PrxY1-xBa2Cu3O7- delta : Inelastic-neutron-scattering, specific-heat, and susceptibility study.
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Journal Article↗
Regulation of calcium mobilization and entry in human platelets by endothelium-derived factors.
Stimulation of Ca2+ mobilization and entry by agonists such as ADP, thrombin, and thromboxane is an early step of platelet activation. Here, we compared the effects of adenosine 3',5'-cyclic monophosphate (cAMP)-elevating prostaglandins, guanosine 3',5'-cyclic monophosphate (cGMP)-elevating nitrovasodilators, membrane-permeant selective activators of cAMP- or cGMP-dependent protein kinases, and physiological endothelium-derived factors on the agonist-evoked Ca2+ mobilization and entry in human platelets. Prostaglandin E1, the prostacyclin analogue Iloprost, the nitric oxide (NO) donor 3-morpholinosydnonimine hydrochloride, and selective activators of cGMP- or cAMP-dependent protein kinase strongly inhibited the agonist-evoked Ca2+ mobilization from intracellular stores and associated late Ca2+ entry but had little effects on the rapid (1st) phase of ADP-evoked Ca2+ entry. During coincubation of platelets with endothelial cells, endothelium-derived factors that were released strongly inhibited platelet agonist-evoked Ca2+ mobilization and only moderately affected the rapid phase of ADP-evoked Ca2+ entry. These effects were partially prevented when endothelial cells were preincubated with cyclooxygenase and/or NO synthase inhibitors. Endothelial cells therefore produce sufficient quantities of labile platelet inhibitors whose effects on the platelet Ca2+ response resemble those observed with selective cAMP- and cGMP-dependent protein kinase activators.
Biological Transport↗
Endothelium-dependent phosphorylation of vasodilator-stimulated protein in platelets during coronary passage.
Compounds that elevate intraplatelet guanosine 3',5'-cyclic monophosphate (cGMP) or adenosine 3',5'-cyclic monophosphate (cAMP) stimulate the phosphorylation of a 46- to 56-kDa thrombocyte protein designated "vasodilator-stimulated protein" (VASP), which is most likely involved in the regulation of adhesion/aggregation. We investigated whether endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) and prostaglandin I2 (PGI2) affected VASP phosphorylation in washed human platelets that were injected into the coronaries of saline-perfused rabbit hearts (n = 22) and collected immediately after passage. The endothelial stimulator acetylcholine (ACh; 1 microM) significantly increased the concentration of cGMP (indicating release of EDRF) and PGI2 in the coronary venous effluent, as well as the concentration of cGMP and cAMP in platelets. Phosphorylation state of VASP increased from 32.1 +/- 2.9 to 64.8 +/- 2.7%. Inhibition of EDRF/NO synthesis by NG-nitro-L-arginine (30 microM) completely abolished the ACh-induced cGMP increase, attenuated the cAMP-elevation without affecting PGI2, and caused a 20.5 +/- 5.8% decrease of the phosphorylation state of VASP. Indomethacin (30 microM) virtually abolished ACh-induced increases of PGI2, cAMP (but not cGMP), and phosphorylated VASP. These results indicate that both EDRF/NO and PGI2 contribute to the regulation of VASP phosphorylation in platelets collected after a single coronary passage. Their synergistic inhibitory effects on platelet function may thus be mediated by a common effect on target proteins like VASP as well as by a secondary increase in cAMP in response to cGMP-elevating compounds such as EDRF.
Acetylcholine↗
Human cyclic GMP-dependent protein kinase I beta overexpression increases phosphorylation of an endogenous focal contact-associated vasodilator-stimulated phosphoprotein without altering the thrombin-evoked calcium response.
The role of the cGMP-dependent protein kinase (cGK) and one of its major substrates, the vasodilator-stimulated phosphoprotein (VASP), in the regulation of a receptor-evoked calcium response was investigated. The human type I beta cGK was stably transfected in human embryonic kidney 293 cells and Swiss mouse 3T6 fibroblasts, which contained significant or no detectable levels of the focal adhesion protein VASP, respectively. Western blot analysis and protein kinase activity measurements demonstrated an 8-fold overexpression of cGK-I beta in 293 cells (7-fold in 3T6 cells), representing an intracellular cGK concentration of 0.33 microM. In experiments with intact 293 cells expressing cGK-I beta, beta-phenyl-1,N2-etheno-cGMP and 8-(p-chlorophenylthio)-cGMP were capable of converting up to 30-40% of the 46-kDa VASP to its 50-kDa phospho- form, equivalent to results observed with cGMP analogs that cause a marked inhibition of the stimulated Ca2+ transient in intact human platelets. In contrast to platelets, preincubation of fura-2-loaded 293 and 3T6 cells with 8-(p-chlorophenylthio)-cGMP did not significantly inhibit thrombin-evoked calcium transients, although sufficient cGK-mediated VASP phosphorylation was clearly detectable under these conditions in cGK-I beta-expressing 293 cells. These results demonstrate that cGK inhibition of agonist-evoked calcium mobilization is not a mechanism common to all cell types and that VASP phosphorylation may not be an essential or sufficient component of the cGK effect on calcium levels. In contrast, the observed VASP phosphorylation mediated by recombinant human cGK-I beta in intact 293 cells does support the hypothesis that focal adhesions and their associated proteins are important cellular sites of cGK action.
Blood Proteins↗
The nitric oxide and cGMP signal transduction system: regulation and mechanism of action.
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Amino Acid Oxidoreductases↗
Defective nitrovasodilator-stimulated protein phosphorylation and calcium regulation in cGMP-dependent protein kinase-deficient human platelets of chronic myelocytic leukemia.
The presence and functional role of the cyclic nucleotide signal transduction system was investigated in platelets from patients with myeloproliferative disorders. Platelets from certain patients with chronic myelocytic leukemia showed decreased expression of cGMP-dependent protein kinase, and platelets from two such patients were studied in some detail. These platelets had very little if any cGMP-dependent protein kinase but a normal level of cAMP-dependent protein kinase. They also contained a normal level of VASP (vasodilator-stimulated phosphoprotein, a specific substrate of both cAMP- and cGMP-dependent protein kinase), as well as a functionally intact prostaglandin E1-stimulated cAMP-mediated VASP phosphorylation. In contrast, sodium nitroprusside-stimulated VASP phosphorylation was severely impaired in these cGMP-dependent protein kinase-deficient platelets, despite an exaggerated cGMP response to sodium nitroprusside. Furthermore, whereas selective activation of the cGMP-dependent protein kinase by 8-(4-chlorophenylthio)-cGMP strongly inhibited the ADP- or thrombin-evoked calcium mobilization from intracellular stores in normal platelets, this agonist-evoked calcium response was not inhibited by the cGMP analog in cGMP-dependent protein kinase-deficient platelets. The results demonstrate a defect in the nitrovasodilator-/cGMP-regulated signal transduction system in human platelets from some patients with myeloproliferative disorders, and underscore that a cGMP-dependent protein kinase regulatory system, distinct from that of cAMP-dependent protein kinase or other cGMP-dependent effectors is operative in normal human platelets.
Blood Platelets↗
Role of cyclic nucleotide-dependent protein kinases and their common substrate VASP in the regulation of human platelets.
The activation of human platelets is inhibited by two intracellular pathways regulated by either cGMP- or cAMP-elevating agents. There is considerable evidence that the inhibitory effects of cGMP and cAMP are mediated by the cGMP-PK and cAMP-PK, respectively, in human platelets. The cGI-PDE is an additional target for cGMP, and the cGMP-mediated elevation of cAMP levels contributes to the well known synergism between cAMP- and cGMP-elevating platelet inhibitors. Stimulation of both cAMP-PK and cGMP-PK prevents the agonist-induced activation of MLCK and PKC and inhibits the agonist-induced calcium mobilization from intracellular stores without any major effect on the ADP-regulated cation channel. These studies suggest that the inhibition of an early event of platelet activation, e.g. activation of PLC, is an effect common to both cGMP-PK and cAMP-PK stimulation. A common substrate of both cGMP-PK and cAMP-PK, the 46/50 kDa protein VASP, has been recently identified as a novel microfilament- and focal contact-associated protein whose phosphorylation correlates very well with platelet inhibition. Future investigations will have to identify the precise molecular mechanism of cyclic nucleotide inhibition of Ca2+ discharge from intracellular stores and whether cGMP-PK- and cAMP-PK-mediated VASP phosphorylation is an important component of this effect of cyclic nucleotides in human platelets.
Adenylyl Cyclases↗
Properties and regulation of human platelet cation channels.
The stimulation of calcium influx by various human platelet agonists which differ in their activation pathways was investigated. ADP activates a receptor-operated cation channel (ROC) and stimulates a phospholipase C (PLC)/inositol-trisphosphate (IP3)-mediated calcium mobilization associated with a secondary calcium influx. Thrombin only stimulates the PLC/IP3-mediated calcium mobilization and associated calcium influx, perhaps followed by an additional phase of calcium influx. The platelet calcium response after incubation with the thromboxane A2 mimetic U 46619 is similar but more transient compared to that after thrombin stimulation. Tert-butylhydroquinone (an inhibitor of endoplasmatic reticulum Ca(2+)-ATPases and cyclooxygenase) elevates cytosolic calcium levels by emptying intracellular calcium stores and stimulates a biphasic calcium influx. Activation of platelet cAMP- and cGMP-dependent protein kinases inhibits the ADP- and thrombin-evoked, calcium store-associated cation influx, but not the fast receptor operated cation influx induced by ADP. Experiments with various ADP-analogs, ATP and ATP-gamma-S suggest that two different ADP-receptors may mediate the calcium responses in human platelets.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Progression of myopathology in Kearns-Sayre syndrome: a morphological follow-up study.
We report on the progression of myopathology by comparing two biopsies from a patient with a Kearns-Sayre-Syndrome. The first biopsy was taken in 1979 and showed 10% ragged-red fibers. Myopathic changes were slight including internal nuclei and fiber splitting in 10% of the fibers. Electron microscopy revealed typical mitochondrial abnormalities with regard to number and shape. In 1989 a second biopsy was performed for an extended analysis of mitochondrial DNA. This time less than 5% of all fibers were ragged-red. Severe myopathic changes could be detected which so far has rarely been reported in mitochondrial cytopathy.
Blotting, Southern↗
The cGMP-dependent protein kinase--gene, protein, and function.
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Amino Acid Sequence↗