Binding of angiotensins to rat brain tissue: structure activity relationship

The binding of 3H-angiotensin II to a synaptosome-enriched fraction of the subcortical part of rat brain was studied. In this fraction specific high-affinity binding sites for angiotensin II were demonstrated. The binding sites were saturated at a ligand concentration of 2 X 10(-9) M. Scatchard analysis revealed a single class of binding sites with an apparent maximal binding capacity of 14 fmoles/mg of protein and an equilibrium dissociation constant, KD, of 0.9 X 10(-9) M. The specific binding at the KD concentration amounted to 59% of the total binding and was reversible. The association and dissociation rate constants (k1 and k-1) were 0.0212 nM-1 min-1 and 0.0196 min-1, respectively. Binding was dependent on both incubation time and tissue concentration in the incubation mixture. Angiotensins with biological activity in the brain, i.e., angiotensins I, II, III, and the fragments (3-8) and (4-8) competed with 3H-angiotensin II for the binding sites with IC50's of 9 X 10(-8) M, 2 X 10(-9) M, 4 X 10(-9) M, 4 X 10(-7) M and 4 X 10(-6), respectively. In the presence of 1 mM of the converting enzyme inhibitor SQ 14,225 the IC50 for angiotensin I was 2 X 10(-7) M. Competition by the biologically active fragment angiotensin (5-8) could not be demonstrated. The latter peptide, however, was highly metabolized during the incubation under the assay conditions used. The binding potency of the various angiotensins paralleled their dipsogenic and pressor potency. The present data indicate the possible physiological involvement of these binding sites as specific receptors in the actions of angiotensins in the brain.