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

Abstract

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 [3H][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 KD of 257 pM. Saturation binding experiments yielded average values of KD = 447 +/- 103 pM, Bmax = 862 +/- 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]-substance P yielded average values of KD = 491 +/- 48 pM and Bmax = 912 +/- 67 fmol/mg of protein. In [3H]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.

Non‐peptide antagonists, CP‐96,345 and RP 67580, distinguish species variants in tachykinin NK1 receptors

“…UCI1 and LRM55 cells have been shown to express NK, receptors (Johnson & Johnson, 1992;Perrone et al, 1986). In order to determine whether other tachykinin receptors are also present on LRM55 and UCIl cells we measured the ability of senktide, a selective NK3 agonist (Wormser et al, 1986), and [PAla8]NKA(4-10), a selective NK2 agonist (Rovero et al, 1989), to stimulate [3H]-inositol phosphate accumulation, as both of these receptor types have been shown to stimulate inositol phospholipid hydrolysis (Bristow et al, 1987;Guard et al, 1988).…”

“…Using the method described by Johnson & Johnson (1992) Lazareno & Roberts (1987). CP-96,345 is the single (2S,3S) enantiomer.…”

“…We have used: UCI1 cells, derived from a human astrocytoma, on which a high density of functional NKI receptors have been characterized by radioligland binding and inositol phosphate formation (Johnson & Johnson, 1992); the rat LRM55 glial cell line which contains large numbers of functional NK, receptors (Perrone et al, 1986) and the well-characterized guinea-pig ileum which expresses both NKI and NK3 receptors (Guard et al, 1988). These cell lines and the tissue have been used to demonstrate that the potency of both non-peptide antagonists, in antagonizing a biochemical response, is species-dependent.…”

Characterization of the endokinins: Human tachykinins with cardiovascular activity

“…Assays were performed with membranes from human glioblastoma astrocytoma UC11 cells (21) 3 receptor incubations were performed in 20 mM Hepes (pH 7.4)͞1 mM MnCl 2 ͞0.1% BSA, all containing 10 M phosphoramidon with or without various concentrations of cold ligand, for 90 min at 27°C in 1-ml assays. Incubations were stopped by rapid filtration through Whatman GF͞C filters, presoaked for 15 min in 0.5% BSA, with a Brandel tissue harvester (Gaithersburg, MD).…”

A Role for Histamine and Substance P in Immediate Allergic Responses in Guinea Pig Airways: Characterization of MDL 108,207DA, a Dual H₁ / NK-1 Receptor Antagonist

“…The ability of putative tachykinin receptor antagonists to inhibit SP-induced increases in phosphatidylinositol turnover in a dose-dependent manner was evaluated in the human astrocytoma NK-1 receptor cell line UC11 [18] as has been described previously [14]. Briefly, cultured cells were loaded with 0.5 ml of 0.2 µ M myo[2- 3 H(N)] inositol (American Radiolabeled Chemicals Inc., specific activity, 20 Ci/mmol) 20–24 h prior to the assay and maintained at 37 °C in a 5% CO 2 environment.…”