Characterization of Receptors for Substance P in Human Astrocytoma Cells: Radioligand Binding and Inositol Phosphate Formation

UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin‐1 receptor ligand [ 3 H][Sar 9 ,Met(O 2 ) 11 ]‐substance P. Kinetic binding experiments conducted at 4°C yielded an association rate constant k 1 of 1.86 X 10 7 M –1 min –1 , a dissociation rate constant k –1 of 0.00478 min –1 , and a calculated kinetic K D of 257 p M . Saturation binding experiments yielded average values of K D = 447 ± 103 p M , B max = 862 ± 93 fmol/mg of protein. This B max corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar 9 ,Met(O 2 ) 11 ]‐substance P yielded average values of K D = 491 ± 48 p M and B max = 912 ± 67 fmol/mg of protein. In [ 3 H]inositol‐labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20‐fold within ∼15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60–120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin‐1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.