Hepatic Effects of Endothelin: Metabolism of [125I]Endothelin-1 by Liver-Derived Cells

Endothelin, a potent vasoactive peptide originally isolated from cultured porcine endothelial cells, (1) elicits hemodynamic and glycogenolytic actions in perfused rat liver; (2) evokes phosphoinositide signaling in hepatic cells; and (3) stimulates synthesis of mediators in Kupffer cells and glucose production in hepatocytes. Recently, we characterized receptor(s) for endothelin on hepatocytes (C. R. Gandhi, R. H. Behal, S. A. K. Harvey, T. Nouchi, and M. S. Olson, Biochem. J. 287, 897-904, 1992). Both hepatocytes and Kupffer cells rapidly internalize [125I]endothelin-1 ([125I]ET-1). In the present study we exposed primary cultures of hepatocytes or Kupifer cells to [125I]ET-1 and analyzed the radiolabeled metabolites which appeared in the cell medium. Six metabolites were separated by high-performance liquid chromatography (HPLC) from hepatocyte medium; these peaks had approximate elution times of 5 (free iodide), 22,35, 37, 38, and 41 min, respectively, whereas the elution time for [125I]ET-1 was 43 min. The kinetics of formation of the metabolites, and experiments using excess unlabeled ET-1, both showed that internalization of the native peptide by hepatocytes is required for the metabolism of [125I]ET-1 into metabolite22, and for the subsequent deiodination of metabolite22. The formation of metabolites35-41 does not require internalization of the native peptide. In Kupffer cells, the cell medium contained only metabolite22 and metabolite41. Internalization of the native peptide was required for the formation of metabolite22 but not for metabolite41. Three classes of [125I]ET-1 metabolites from hepatic cells also were separated by sequential precipitation with trichloroacetic acid (TCA) and with silver nitrate. This procedure facilitated multiple rapid assays of [125I]ET-1 metabolism. Phosphoramidon, an inhibitor of neutral metalloendopeptidases, did not affect signiticantly the binding or the metabolism of [125I]ET-1 by hepatocytes or Kupffer cells. The aminopeptidase inhibitor bacitracin strongly attenuated [125I]ET-1 metabolism by hepatocytes, with a concomitant increase in the intracellular content of [125I]ET-1. These data suggest that enzymes capable of endothelin degradation are present both on the surface and in the intracellular compartment of hepatic cells, and that endothelin is not metabolized by neutral endopeptidase 24.11 in the liver.