Lu X Y, Zhang W, Grove K L, Speth R C
Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman 99164-6520, USA.
J Pharmacol Toxicol Methods. 1995 Apr;33(2):83-90. doi: 10.1016/1056-8719(94)00061-8.
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.
评估血管紧张素II（AII）受体结合通常需要在检测放射性配体结合之前将感兴趣的组织冷冻。本研究评估了以不同速率冷冻各种大鼠组织对125I-肌氨酸1、异亮氨酸8血管紧张素II（125I-SI AII）与AII受体亚型结合的影响。在-20℃隔层中缓慢冷冻显著降低了125I-SI AII与肾上腺（51%）、附睾（34%）和肝脏（22%）中AT1受体的结合。125I-SI AII与脑中AT1受体的结合未显著降低。在肾上腺中，冷冻使AT1受体的Bmax和亲和力均降低。但在附睾中，仅AT1受体的亲和力降低。冷冻也未显著降低125I-SI AII与肾上腺、附睾和脑中AT2受体的结合。对肾上腺中125I-SI AII与AT1受体结合减少机制的进一步评估表明，冷冻使受体密度和对125I-SI AII的亲和力均降低。在干冰浴中快速冷冻导致125I-SI AII与肾上腺中AT1受体的结合减少得更多。在液氮中速冻使肾上腺中125I-SI AII的结合减少程度与缓慢冷冻相似。这些结果表明，涉及组织冷冻的AII受体亚型研究低估了AT1受体亚型的密度和亲和力。
