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Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin

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Author: Draijer, R. · Vaandrager, A.B. · Nolte, C. · Jonge, H.R. de · Walter, U. · Hinsbergh, V.W.M. van
Institution: Gaubius Instituut TNO
Source:Circulation Research, 5, 77, 897-905
Identifier: 233055
doi: DOI:10.1161/?01.RES.77.5.897
Keywords: Biology · aortic endothelial cells · cGMP · endothelial contraction · microvascular endothelial cells · permeability · 8 (4 chlorophenylthio) cyclic GMP · calcium ion · cyclic GMP · cyclic GMP derivative · fura 2 · horseradish peroxidase · phosphoprotein · protein kinase · thrombin · unclassified drug · vasodilator stimulated phosphoprotein · aorta · article · calcium cell level · cell membrane permeability · endothelium cell · human · human cell · iliac artery · iliac vein · prepuce · priority journal · protein phosphorylation · umbilical vein · vascular endothelium · Aorta · Autoradiography · Blotting, Northern · Calcium Channels · Capillary Permeability · Cell Adhesion Molecules · Cells, Cultured · Comparative Study · Cyclic GMP · Cyclic GMP-Dependent Protein Kinases · Cytoplasm · Endothelium, Vascular · Enzyme Activation · Hemostasis · Human · Iliac Artery · Iliac Vein · Immunohistochemistry · Microcirculation · Microfilament Proteins · Models, Biological · Phosphoproteins · Phosphorylation · Platelet Aggregation Inhibitors · RNA · Support, Non-U.S. Gov't · Thionucleotides · Thrombin · Time Factors · Umbilical Veins


Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT- cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin- stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca2+](i) was strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca2+ and endothelial permeability. These data indicate that cGMP-PK type I is expressed in various types of human macrovascular and microvascular ECs but is absent or expressed in very low amounts in umbilical vein ECs. cGMP-PK type I expression in ECs may be important in the regulation of endothelial permeability and the release of factors involved in vasoregulation and hemostasis. Chemicals/CAS: 8-((4-chlorophenyl)thio)cyclic-3',5'-GMP, 54364-02-2; Calcium Channels; Cell Adhesion Molecules; Cyclic GMP, 7665-99-8; Cyclic GMP-Dependent Protein Kinases, EC; Microfilament Proteins; Phosphoproteins; Platelet Aggregation Inhibitors; RNA, 63231-63-0; Thionucleotides; Thrombin, EC; vasodilator-stimulated phosphoprotein