Expression of cGMP-Dependent Protein Kinase I and Phosphorylation of Its Substrate, Vasodilator-Stimulated Phosphoprotein, in Human Endothelial Cells of Different Origin

• Published in: Circulation research 1995-11, Vol.77 (5), p.897-905
• Main Authors: Draijer, Richard, Vaandrager, Arie B, Nolte, Christine, de Jonge, Hugo R, Walter, Ulrich, van Hinsbergh, Victor W.M
• Format: Article
• Language: English
• Subjects: Aorta; Autoradiography; Biological and medical sciences; Blood vessels and receptors; Blotting, Northern; Calcium Channels - drug effects; Calcium Channels - metabolism; Capillary Permeability; Cell Adhesion Molecules - metabolism; Cells, Cultured; Cyclic GMP - analogs & derivatives; Cyclic GMP - pharmacology; Cyclic GMP-Dependent Protein Kinases - genetics; Cyclic GMP-Dependent Protein Kinases - metabolism; Cytoplasm - drug effects; Cytoplasm - metabolism; Endothelium, Vascular - cytology; Endothelium, Vascular - enzymology; Endothelium, Vascular - metabolism; Enzyme Activation; Fundamental and applied biological sciences. Psychology; Hemostasis; Humans; Iliac Artery; Iliac Vein; Immunohistochemistry; Microcirculation; Microfilament Proteins - metabolism; Models, Biological; Phosphoproteins - metabolism; Phosphorylation; Platelet Aggregation Inhibitors - pharmacology; RNA - isolation & purification; Thionucleotides - pharmacology; Thrombin - pharmacology; Time Factors; Umbilical Veins; Vertebrates: cardiovascular system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.res.77.5.897

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 [Ca plus]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 Ca plus 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.(Circ Res. 1995;77:897-906.)