Endothelin association with the cultured rat Kupffer cell: Characterization and regulation

Abstract

Circulating endothelin (ET) levels are elevated in conditions such as endotoxemia, hepatic ischemia-reperfusion injury, or orthotopic liver transplantation, and this potent peptide may contribute to hepatic pathophysiology. We measured the surface binding of [125I]ET-1 to rat Kupffer cells in primary culture at 4 degrees C; the dissociation constant (Kd) was 270 pmol/L, and the apparent Bmax was 3,000 receptors/cell. At 37 degrees C, total association (surface binding plus internalization) was much greater than at 4 degrees C, indicating that internalization of the receptor-ligand complex is rapid; the apparent Kd was 30 pmol/L, comparable with other reports for hepatic-derived cells. Studies using [125I]ET-1, [125I]ET-3, and specific ET (ant)agonists showed that Kupffer cells possess predominantly ET(B) type receptors. Prior treatment with 500 pmol/L unlabeled endothelin rapidly ( < 15 minutes) occluded 60% of subsequent [125I]ET association; using 5 nmol/L unlabeled ET, this occlusion occurred within 1 minute. [125I]ET association with Kupffer cells was unaffected by short-term (approximately 1 hour) treatment with cyclic adenosine monophosphate (cAMP), but long-term (20 hour) treatment resulted in a twofold increase in [125I]ET association with no change in the apparent Kd. Stimulation of protein kinase C in Kupffer cells by phorbol 12-myristate acetate had a dual regulatory effect on [125I]ET association. Short-term ( < 1 hour) treatment with phorbol 12-myristate acetate decreased [125I]ET-3 association by 50%, whereas prolonged treatment (20 hour) increased association twofold. In both cases, the apparent Kd for [125I]-endothelin was unaltered.