SPECIFIC BINDING-SITES FOR (3-8)ANGIOTENSIN IN C6 GLIOMA-CELLS

A novel putative receptor for (3-8)angiotensin (AngIV) has recently be en found in various tissues, including the brain. However, the localiz ation 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 lin e (C6) and in rat primary glial cells. Using [I-125]AngIV as a radioli gand, 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 mu M(- 1) and a density of 563 +/- 190 fmol/mg protein. The ligand specificit y of the AngIV sites was determined from competitive displacement assa ys with AngIV, AngIII, (4-8)AngII, [Sar(1),Ile(8)]-AngII, losartan (an angiotensin subtype 1 receptor ligand) and PD123319 (an angiotensin s ubtype 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 mu M. The ra dioreceptor assay data were confirmed by receptor autoradiography on c ells grown on glass slides. Moreover, the bound radioligand was shown by HPLC to be [I-125]AngIV and not a breakdown product. Preliminary ex periments with primary astrocyte cultures showed the presence of AngIV binding sites. It was concluded that C6 glioma cells and primary glia l cells have AngIV binding sites with properties like those reported f or the whole brain and distinct from those of AT receptors.