Internalization of endothelin by cultured human vascular smooth muscle cells: characterization and physiological significance.
The binding and internalization of 125I-endothelin (125I-ET-1) was studied in cultured human vascular smooth muscle cells (hVSMC). Discrimination between surface-bound and internalized radiolabeled ligand was achieved using either acetic acid or trypsin treatment of cell layers, with the two procedures yielding comparable results. Total cellular 125I-ET-1 binding hVSMC at 37 degrees was rapid and reached near equilibrium within 30 min. Such binding could be resolved into surface-bound (acid/trypsin-sensitive) and internalized (acid/trypsin-resistant) components. The accumulation of internalized 125I-ET-1 was temperature dependent and occurred at 37 degrees (t1/2 approximately 15 min) but not at 4 degrees. Internalization of 125I-ET-1 by hVSMC was reversibly inhibited by the transglutaminase inhibitor dansylcadaverine (half-maximal inhibitory concentration, approximately 400 microM). Cytosolic acidification of hVSMC (from pH approximately 6.8 to approximately 6.3) by incubation with potassium acetate in a choline buffer also inhibited 125I-ET-1 internalization. Our observation indicate that smooth muscle cells internalize ET-1 via the clathrin-mediated endocytotic pathway. Dansylcadaverine and other inhibitors of transglutaminase inhibited ET-1-stimulated inositol phospholipid hydrolysis in hVSMC and decreased ET-1-induced vasoconstriction in isolated endothelium-denuded blood vessels. Internalization of ET-1 may, therefore, be relevant to the characteristically protracted physiological effects of this peptide on the vasculature.
Department of Research, University Hospital, Basel, Switzerland.
T J
TJ
Resink
T
T
Scott-Burden
C
C
Boulanger
E
E
Weber
F R
FR
Bühler
D002103:Cadaverine / Q000031:analogs & derivatives / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D003331:Coronary Vessels; D003600:Cytosol / Q000378:metabolism; D016232:Endothelins; D004730:Endothelium, Vascular / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism* / Q000493:pharmacokinetics; D013696:Temperature
D016232:Endothelins; D010455:Peptides; C008542:monodansylcadaverine; D002103:Cadaverine