Endothelin and nitric oxide synthase in lymphatic endothelial cells: Immunolocalization in vivo and in vitro

Abstract

Background: Endothelin (ET) is an endothelium-derived multifunctional peptide that produces a potent, long-lasting vasoconstriction. Nitric oxide (NO), besides being the most important endothelium-derived relaxant factor in blood vessels, is supposed to be involved in regulating the interactions among endothelium, adhesive molecules, and leukocytes. Methods: We investigated the possible occurrence and distribution of ET and constitutive nitric oxide synthase (NOs), the enzyme that generates NO from L-arginine, in bovine lymphatic vessels and primary culture of lymphatic endothelium by using immunocytochemistry. Results: Specific immunostaining with both ET and endothelial constitutive NOs antisera was detectable at light and electron microscopic levels in the endothelial cell layer of lymphatic vessels, whereas no immunostaining could be observed in the muscular and adventitial layers. The immunoreaction showed a diffuse pattern throughout the cytoplasm of endothelial cells. Primary cultures of endothelial cells isolated from lymphatic vessels also displayed cytoplasmic ET- and NOs-like immunoreactivities. The endothelial nature of cell monolayers was confirmed by the positive reaction to the yon Willebrand factor, a reliable marker of endothelial cells, and by ultrastructural features of cultured cells. Conclusions: These findings suggest that the endothelium is a major source of ET and NO in lymphatic vessels. Interestingly, the lymphatic endothelium maintains the capability of producing such vasoactive sustances also in vitro, thus suggesting that lymphatic endothelial cells in culture may be used in studies concerning the role of the endothelium in the generation of vasoactive molecules. According to previous functional studies, the occurrence of ET and NOs immunoreactivities in lymphatic vessel endothelium supports the view that lymphatic endothelium may play an important role in the regulation of lymphatic vascular tone and in the production of vascular contractile activity promoting lymph flow.