Characterization of two angiotensin II binding sites in cultured mouse spinal cord neurones

Characteristics of angiotensin II (AII) binding have been determined in cultured mouse spinal cord neurones using [125I]AII and [3H]AII. The Scatchard plot of equilibrium binding was curvilinear and could be described by postulating the existence of two different classes of independent binding sites (Kd1 = 0.43 nM, Bmax1 = 12.5 fmol/1.5 X 10(6) cells; Kd2 = 25.6 nM, Bmax2 = 220 fmol/1.5 X 10(6) cells). These values are in close agreement with the Kd values obtained from kinetic studies. The high affinity binding sites appeared to be similar to the single class of sites described in other studies. The relative inhibition potency of AII-related peptides was studied. Sar1,-Leu8-AII was the most potent in inhibiting specific AII binding. The characteristics of the two AII binding sites suggest that they correspond to two receptors as described in a previous electrophysiological approach using this model in our laboratory. Taken together, these data confirm that this model of neurones in primary culture is a unique and very attractive model of receptor studies. The classical criteria necessary for positive identification of a ligand-receptor have been satisfied: saturability, reversibility, specificity and most importantly correlation of the binding parameters and biological effects of AII.