K. Stephenson et al., ENDOTHELIN ASSOCIATION WITH THE CULTURED RAT KUPFFER CELL - CHARACTERIZATION AND REGULATION, Hepatology, 22(3), 1995, pp. 896-905
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.