Characterization of angiotensin II binding sites on neuroblastoma × glioma hybrid cells

Abstract

Mouse neuroblastoma×rat glioma hybrid cells, NG108-15, have recently been shown to contain immunoreactive angiotensin II (AII). In the present study, we have examined this hybrid cell line for the presence of specific AII binding sites using [ 125I] AII. Specific AII binding was saturable and reversible. Scatchard analysis revealed a linear plot with an affinity constant (K d) of 0.323 nM and a binding capacity (B max) of 7.13 fmol/mg protein. Kinetic studies demonstrated an association rate constant (K +1) of 3.55 × 10 6 M −1 sec −1 and a dissociation rate constant (K −1) of 4.18× 10 −4 sec −1. Displacement curves, using concentrations of 10 −11 M to 10 −4 M of unlabeled AII, revealed high and low affinity components of the AII binding site with IC 50's of 0.46 nM and 1.75 μm respectively. The AII antagonist, saralasin, had approximately equal potency with unlabeled AII at the high affinity site. Furthermore, structurally related and unrelated peptides had no significant inhibitory effect on AII binding. This study demonstrates that specific AII binding sites are present on NG108-15 cells, and that these binding sites are similar in kinetic character to the AII receptor that has been previously identified in membranes from mammalian brain. It is concluded that the NG108-15 hybrid cells may provide a useful continuous cell line model for investigating both the biochemical and molecular properites of the AII binding site.