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°C compartment significantly reduced ¹²⁵I-SI AII binding to AT₁ receptors in the adrenal (51%), epididymis (34%), and liver (22%). Binding of ¹²⁵I-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 receptor subtypes that involve freezing of the tissues underestimate the density and affinity of the AT₁ receptor subtype.