EXPRESSION OF CGMP-DEPENDENT PROTEIN-KINASE-I AND PHOSPHORYLATION OF ITS SUBSTRATE, VASODILATOR-STIMULATED PHOSPHOPROTEIN, IN HUMAN ENDOTHELIAL-CELLS OF DIFFERENT ORIGIN

Previous studies demonstrated that the thrombin-induced permeability o f 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 endotheli al cells (ECs) and the role of cGMP-PK in the reduction of thrombin-in duced endothelial permeability was investigated. cGMP-PK type I was de monstrated in freshly isolated ECs from human aorta and iliac artery a s well as in cultured ECs from human aorta, iliac vein, and foreskin m icrovessels. Addition of the selective cGMP-PK activator 8-(4-chloroph enylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of th e 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 mono layers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimula ted permeability, as determined by measuring the peroxidase passage th rough EC monolayers on porous filters. Furthermore, the thrombin-induc ed rise in cytoplasmic [Ca2+](i) was strongly attenuated by the cGMP-P K 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 increas es in cytoplasmic Ca2+ and endothelial permeability. These data indica te that cGMP-PK type I is expressed in various types of human macrovas cular and microvascular ECs but is absent or expressed in very low amo unts in umbilical vein ECs. cGMP-PK type I expression in ECs may be im portant in the regulation of endothelial permeability and the release of factors involved in vasoregulation and hemostasis.