Influence of tissue freezing on the binding of 125I-sarcosine1, isoleucine8 angiotensin II to angiotensin II receptor subtypes in the rat.

Evaluation of angiotensin II (AII) receptor binding often necessitates freezing of the tissue of interest prior to assay of radioligand binding. This study evaluated the effects of freezing of various rat tissues at different rates on 125I-sarcosine1, isoleucine8 angiotensin II (125I-SI AII) binding to AII receptor subtypes. Slow freezing in a -20 degrees C compartment significantly reduced 125I-SI AII binding to AT1 receptors in the adrenal (51%), epididymis (34%), and liver (22%). Binding of 12tI-SI AII to the AT1 receptor in the brain was not significantly reduced. In the adrenal, both the Bmax and affinity of AT1 receptors were decreased by freezing. But in the epididymis, only the affinity of AT1 receptors was decreased. Binding of 125I-SI AII to AT2 receptors in the adrenal, epididymis, and brain was also not significantly reduced by freezing. Further evaluation of the mechanism of the reduction in 125I-SI AII binding to AT1 receptors in the adrenal indicated that both the receptor density and affinity for 125I-SI AII were decreased by freezing. Rapid freezing in a dry-ice bath caused even greater reductions in 125I-SI AII binding to AT1 receptors in the adrenal. Snap freezing in liquid nitrogen decreased 125I-SI AII binding in adrenals to a similar extent as did slow freezing. These results suggest that studies of AII receptors subtypes that involve freezing of the tissues underestimate the density and affinity of the AT1 receptor subtype.