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Inhibition of proteolytic degradation and characterization of binding of natural angiotensins to AT1 receptors in brain and liver

Metabolism of angiotensins in receptor binding assays is a continuing problem. The suggestion that brain AT1 receptors (AT1R) differ from peripheral AT1R necessitates accurate appraisal of brain AT1R binding. The goal of this study was to establish experimental conditions that preserve 125I‐Ang II and ‐III, to characterize their binding to rat hypothalamic and liver AT1R. Negligible specific binding of 125I‐Ang III and ‐II is observed in hypothalamic membranes (45 or 60min at 24oC in 5mM EDTA, 150mM NaCl, 0.1mM bacitracin, 50mM NaPO4, pH 7.2 buffer + 10 μM PD123319) due to severe proteolytic degradation, as < 5% of bound 125iodine corresponds to 125I‐Ang III or ‐II. In liver membranes, 125I‐Ang III is severely degraded, whereas 125I‐Ang II shows high affinity saturable binding to AT1R with > 50% of bound 125I corresponding to 125I‐Ang II. Addition of 1 mM o‐phenanthroline & phenylmethylsulfonyl fluoride, and 3 mM puromycin & glutamate phosphonate to the assay buffer increased bound 125I‐Ang II and ‐III to > 80%, both in liver and hypothalamic membranes revealing abundant, high‐affinity binding to AT1R. The affinity of both ligands for brain AT1R was less than for liver. 125I‐Ang III showed a smaller Bmax for AT1R in liver than 125I‐Ang II. These results indicate that both peptides bind with higher affinity for liver AT1R than brain AT1R, but that 125I‐Ang II binds to a larger population of AT1R in liver than does 125I‐Ang III.