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 ( K d1 = 0.43nM,B max1 = 12.5fmol/1.5 × 10 6cells; K d2 = 25.6nM,B max2 = 220fmol/1.5 × 10 6cells). These values are in close agreement with the K d 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 studies. Sar 1, -Leu 8-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 on 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.