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 [H-3][Sar9,Met(O2)11]-substance P. Kinetic binding experiments conducted at 4-degrees-C yielded an association rate constant k1 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 pM. Saturation binding experiments yielded average values of K(D) = 447 +/- 103 pM, 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 [Sar9,Met(O2)11]-substance P yielded average values of K(D) = 491 +/- 48 pM and B(max) = 912 +/- 67 fmol/mg of protein. In [H-3]inositol-labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20-fold within approximately 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.