Specific binding sites for (3–8) angiotensin in C6 glioma cells

Abstract

A novel putative receptor for (3–8)angiotensin (AngIV) has recently been found in various tissues, including the brain. However, the localization of AngIV binding sites to specific types of brain cells has yet to be established. In this study tissue culture was used to determine the presence and characteristics of AngIV binding in a glioma cell line (C6) and in rat primary glial cells. Using [ 125I]AngIV as a radioligand, C6 glioma cells were found by radioreceptor assay to bind with a high affinity and in a saturable, reversible manner. The best fit to the data was for a two-binding-site model; a higher-affinity site with a K a of 2.49 ± 0.46 nM −1 and a density of 33.71 ± 7.8 fmol/mg protein, and a second low-affinity site with a K a of 176 ± 7 gmM −1 and a density of 563 ± 190 fmol/mg protein. The ligand specificity of the AngIV sites was determined from competitive displacement assays with AngIV, AngIII, (4–8)AngII, [Sar 1,IIe 8]-AngII, losartan (an angiotensin subtype 1 receptor ligand) and PD123319 (an angiotensin subtype 2 receptor ligand). The relative order of binding affinity was AngIV > AngIII ≫ (4–8)AngII, while losartan, PD123319 and Sar 1,Ile 8-AngII failed to compete for the AngIV sites, even at 1 μM. The radioreceptor assay data were confirmed by receptor autoradiography on cells grown on glass slides. Moreover, the bound radioligand was shown by HPLC to be [ 125I]AngIV and not a breakdown product. Preliminary experiments with primary astrocyte cultures showed the presence of AngIV binding sites. It was concluded that C6 glioma cells and primary glial cells have AngIV binding sites with properties like those reported for the whole brain and distinct from those of AT receptors.