Specific Binding of Endothelin on Human Bronchial Smooth Muscle Cells in Culture and Secretion of Endothelin-like Material from Bronchial Epithelial Cells

Abstract

Endothelin, synthesized by endothelial cells, is the most potent vasoconstrictor and bronchoconstrictor agent known. We investigated endothelin release from human bronchial epithelial cells and the binding of the peptide to autologous bronchial smooth muscle cells in culture. Epithelial and smooth muscle cells were isolated by enzymatic digestion of bronchial tissue obtained on surgery, and cultured to confluency by standard methods. Epithelial cells stained positively for cytokeratin filaments. Smooth muscle cells stained uniformly for alpha-smooth muscle actin. Immunoreactive endothelin contents in the supernatants of epithelial cells extracted on C8 Amprep columns were evaluated by radioimmunoassay. Epithelial cells released appreciable amounts of immunoreactive endothelin into the culture medium (from 0.65 to 2.1 pmol/ml). A single specific binding site for [125I]endothelin 1 was identified on bronchial smooth muscle cells with an apparent Kd of 113 pM and a maximal binding capacity of 22.1 fmol/10(6) cells. At room temperature the binding was saturable, reached equilibrium at 120 min (25 pM endothelin 1), and was slowly and incompletely reversed by unlabeled endothelin over a period of 8 h. Conditioned medium from epithelial cells inhibited the [125I]endothelin 1 binding, dose dependently, and the effect was antagonized by monospecific antiserum. Thus, human bronchial smooth muscle cells possess specific binding sites for endothelin 1 and human bronchial epithelial cells secrete an endothelin-like material. This may have a role in the pathogenesis of asthma.