ENDOTHELIN ASSOCIATION WITH THE CULTURED RAT KUPFFER CELL - CHARACTERIZATION AND REGULATION

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 pat hophysiology. We measured the surface binding of [I-125]ET-1 to rat Ku pffer cells in primary culture at 4 degrees C; the apparent dissociati on constant (K-d) was 270 pmol/L, and the apparent B-max was 3,000 rec eptors/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 K-d was 30 pmol/L, comparable with other reports for hepatic-derived cells. Studies using [I-125]ET-1, [I-125]ET-3, and specific ET (ant)ag onists showed that Kupffer cells possess predominantly ET(B) type rece ptors. Prior treatment with 500 pmol/L unlabeled endothelin rapidly (< 15 minutes) occluded 60% of subsequent [I-125]ET association; using 5 nmol/L unlabeled ET, this occlusion occurred within 1 minute. [(125)]E T association with Kupffer cells was unaffected by short-term (approxi mate to 1 hour) treatment with cyclic adenosine monophosphate (cAMP), but long-term (20 hour) treatment resulted in a twofold increase in [I -125]ET association with no change in the apparent K-d. Stimulation of protein kinase C in Kupffer cells by phorbol 12-myristate acetate had a dual regulatory effect on [I-125]ET association. Short-term (<1 hou r) treatment with phorbol 12-myristate acetate decreased [I-125]ET-3 a ssociation by 50%, whereas prolonged treatment (20 hour) increased ass ociation twofold. In both cases, the apparent K-d for [I-125]-endothel in was unaltered.