Rat brain cells in primary culture: Characterization of angiotensin II binding sites

Abstract

The binding kinetics of angiotensin II (ANG II) have been studied in primary cultures from fetal rat brain. Binding of [125I]ANG II to rat brain cells in culture is time-, pH- and cell concentration-dependent. The binding is saturable, reversible, and 90--95% specific. Binding follows first-order kinetics, with values for K1 and K-1 of 4.9 x 10(6)M-1 S-1 and 3.33 x 10(4)S-1 respectively. Scatchard analysis reveals the presence of a single class of binding sites with Ka of 1.0 x 10(9)M-1 and an average of approximately 6 x 10(3) sites per cell. [125I]ANG II recovered from incubation medium under the conditions of the binding assay or after dissociation from cells is not significantly degraded as judged by gel filtration on Sephadex G-25 and radioreceptor assay. ANG II analogs compete with [125I]ANG II for binding, with potencies in general paralleling previously established biological activities. Of 5 analogs tested, (Ile8)-ANG II was almost equipotent with ANG II while (Dval3)-ANG II was least potent in the competitive binding assay. These data fulfill criteria for the identification of specific angiotensin II receptors in cells from mammalian brain.