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It is not yet clear whether cellular junctions between splenic sinus endothelial cells are open or closed. In order to clarify this, immunolocalization of thrombomodulin (TM), endothelial protein C receptor (EPCR), protease-activated receptor 1 (PAR1), sphingosine 1-phosphate receptor 1 (S1P1), β-catenin phosphorylated at Try142 (β-catenin Y142) and β-catenin phosphorylated at Try654 (β-catenin Y654), which are related proteins that regulate dissociation and association of the adherens junctions of endothelial cells, are examined in rats using laser microscopy and electron microscopy. TM, EPCR, PAR1 and S1P1 were colocalized in the entire circumference of the endothelial cells, as well as in the caveolar membranes and junctional membranes of adjacent endothelial cells. These molecules may protect the adherens junctions of the endothelial cells. On the other hand, β-catenin Y142 and β-catenin Y 654 colocalized with α-catenin and β-catenin, respectively and in addition, β-catenin Y142 and β-catenin Y 654 were localized in the vicinity of the adherens junctions of the endothelial cells from immunogold electron microcopy. The adherens junctions are considered to be partially dissociated at the site where β-catenin Y142 and β-catenin Y 654 are localized. Thus, the system that protects the adherens junctions and the system that dissociates them may concurrently coexist in the endothelial cells and dissociation and association of the adherens junctions may be constantly repeated at the cell boundary of the endothelial cells. [ABSTRACT FROM AUTHOR]

Localization of integrins β1-8, α1, α2, α3, α5, α6 and αv in sinus endothelial cells of the rat spleen was examined by immunofluorescence microscopy. Labeling for anti-integrin β5 and integrin αv was detected and colocalized in the entire circumference of endothelial cells. Labeling for integrin β5, vinculin and actin filaments demonstrated that they lay close to each other in the basal part of the endothelial cells. Although the other integrin βs, including integrin β1 and integrins α1, α2, α3, α5 and α6 in combination with integrin β1, were localized in leukocytes, slightly large cells, megakaryocytes and/or platelets in the sinus lumen and splenic cords, they were not detected in endothelial cells. Labeling for vitronectin, a component of the extracellular-matrix-binding integrin αvβ5, was strongly stained in the periphery of the wall of sinuses, as was collagen IV and, in addition, was localized in the cytoplasm of endothelial cells. Ultrastructural localization of integrin β5, vitronectin and clathrin was examined by immunogold electron microscopy to elucidate the involvement of integrin αvβ5 in the endocytosis of vitronectin in sinus endothelial cells. Electron microscopy with detergent extraction revealed abundant coated pits and coated vesicles in endothelial cells. Immunogold labeling for vitronectin was present in pits, vesicles and the stacked endoplasmic reticulum. Double-labeling for integrin β5 or integrin αv and clathrin revealed that they were colocalized in some vesicles in close proximity to the apical and lateral plasma membrane of the endothelial cells. The possible functional roles of integrin αvβ5 in endothelial cells of the splenic sinus are discussed. [ABSTRACT FROM AUTHOR]

Localization of three P2X and six P2Y receptors in sinus endothelial cells of the rat spleen was examined by immunofluorescent microscopy, and ultrastructural localization of the detected receptors was examined by immunogold electron microscopy. In immunofluorescent microscopy, labeling for anti-P2Y1, P2Y6, and P2Y12 receptors was detected in endothelial cells, but P2X1, P2X2, P2X4, P2Y2, P2Y4, and P2Y13 receptors was not detected. P2Y1 and P2Y12 receptors were prominently localized in the basal parts of endothelial cells. P2Y6 receptor was not only predominantly localized in the basal parts of endothelial cells, but also in the superficial layer. Triple immunofluorescent staining for a combination of two P2Y receptors and actin filaments showed that P2Y1, P2Y6, and P2Y12 receptors were individually localized in endothelial cells. Phospholipase C-β3, phospholipase C- γ2, and inositol-1,4,5-trisphosphate receptors, related to the release of the intracellular Ca from the endoplasmic reticulum, were also predominantly localized in the basal parts of endothelial cells. In immunogold electron microscopy, labeling for P2Y1, P2Y6, and P2Y12 receptors were predominantly localized in the basal part of endothelial cells and, in addition, in the junctional membrane, basal plasma membrane, and caveolae in the basal part of endothelial cells. Labeling for phospholipase C-β3 and phospholipase C-γ2 was dominantly localized in the basal parts and in close proximity to the plasma membranes of endothelial cells. The possible functional roles of these P2Y receptors in splenic sinus endothelial cells are discussed. [ABSTRACT FROM AUTHOR]