Internalization of [3H]Substance P Analogues in NK-1 Receptor Transfected CHO Cells

Abstract

The internalization of [3H]propionyl[Met(O2)11]SP(7–11) which binds one binding site and of [3H][Pro9]SP which binds the two binding sites associated with the NK-1 receptor has been examined in CHO cells. The quantity of [3H][Pro9]SP measured inside the cytoplasm in kinetic experiments is fully temperature-dependent. In contrast, [3H]propionyl[Met(O2)11]SP(7–11) internalization reaches the same extent whatever the temperature, although the rate slowed down with lower temperature. The extent of internalization of [3H][Pro9]SP relative to the total specific bound is biphasic, when the extent of internalization of [3H]propionyl[Met(O2)11]SP(7–11) remains constant. For [3H][Pro9]SP, a high-affinity high-yield component inhibited in the presence of propionyl[Met(O2)11]SP(7–11) and a low-affinity low-yield component in the internalization process could be determined. Saturation studies show that [3H][Pro9]SP-binding parameters are insensitive to both phenylarsine oxide and monensin treatment, whereas [3H]propionyl[Met(O2)11]SP(7–11) maximal binding is decreased in both cases. Altogether, these data suggest that the two radiolabeled peptides should not follow the same internalization pathway.