IDENTIFICATION AND CHARACTERIZATION OF ANGIOTENSIN-IV BINDING-SITES IN RAT NEURON AND ASTROCYTE CELL-CULTURES

This study demonstrates the existence of the putative receptor for the hexapeptide (3-8) fragment of angiotensin II (AngIV) on rat astrocyte s and neurons grown in cell culture, Binding of I-125-AngIV was satura ble and distinct from that of the AngII receptor subtypes. Equilibrium binding was attained in 15 min in astrocytes and 75 min in neurons at 22 degrees C. The bound peptide was confirmed by HPLC to be intact An gIV while the bound peptide was substantially degraded, even in the pr esence of peptidase inhibitors. Scatchard analysis of equilibrium bind ing was consistent with a two binding site model, revealing a high aff inity and a low affinity binding site in both cell types. In neurons, the respective association constants (Ka) were 2.72 +/- 0.23 nM(-1) an d 727 +/- 354 nM(-1), with associated receptor densities of 109.30 +/- 58.87 and 1723 +/- 1167 fmol/mg protein. Similar analyses in astrocyt es gave Kas of 5.71 +/- 2.85 nM(-1) and 277 +/- 205 nM(-1), and respec tive densities of 191.1 +/- 90.1 and 1425 +/- 1250 fmol/mg protein, Ho wever, the quantitative reliability of these binding isotherms may be influenced by the degration of unbound peptide. Competitive binding an alysis was used to determine the specificity of the receptor site, wit h the relative order of affinities being AngIV>AngIII>AngII(4-8), and no displacement by AngII, losartan and PD123319 in either neurons or a strocytes. Autoradiography with I-125-AngIV performed on neuronal cult ures demonstrated that binding was confined to a subpopulation of the total cells, These data support the existence of a specific binding si te for AngIV in both neurons and astrocytes, consistent with the prope rties of binding reported previously in the brain, and distinguish thi s site from the AngII receptor subtypes.