Smith R D
Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, UK.
Biochem Mol Biol Int. 1994 Apr;32(5):923-31.
The relative binding affinities of non-peptide antagonists, and the sensitivity of 125I-angiotensin II (125I-AII) binding to the reducing agent, dithiothreitol, indicated the presence of AT1 angiotensin receptors on RIE-1 rat intestinal epithelial cells. Consistent with this finding, AT1 angiotensin receptor mRNA was detected in RIE-1 cells using Northern blotting, whereas no mas transcripts (which also encode an angiotensin receptor) were detectable using a RNAse protection assay. At 37 degrees C, 125I-AII rapidly bound to RIE-1 cells and internalised into an acid-inaccessible compartment, which resulted in the depletion of 125I-AII from the binding medium. Following overnight incubation of RIE-1 cells with 125I-AII at 4 degrees C, the majority of bound ligand was also, unexpectedly, found to be inaccessible to subsequent acid elution. After warming these cultures to 37 degrees C, acid-inaccessible 125I-radioactivity rapidly disappeared from the cells, and 125I-labelled degradation products accumulated in the medium. Scatchard analysis of the concentration-dependent binding of 125I-AII solely to the acid-accessible sites on RIE-1 cells revealed little difference to the KD value obtained from total 125I-AII binding (to both acid-accessible and -inaccessible sites), but only approximately 15% of the number of sites.
非肽拮抗剂的相对结合亲和力，以及125I-血管紧张素II（125I-AII）与还原剂二硫苏糖醇结合的敏感性，表明RIE-1大鼠肠上皮细胞上存在AT1血管紧张素受体。与这一发现一致，使用Northern印迹法在RIE-1细胞中检测到了AT1血管紧张素受体mRNA，而使用RNA酶保护试验未检测到mas转录本（其也编码一种血管紧张素受体）。在37℃时，125I-AII迅速与RIE-1细胞结合并内化到一个酸不可进入的区室中，这导致结合介质中125I-AII的消耗。在4℃下将RIE-1细胞与125I-AII过夜孵育后，意外地发现大多数结合的配体也无法被随后的酸洗脱。将这些培养物升温至37℃后，细胞中酸不可进入的125I放射性迅速消失，125I标记的降解产物在培养基中积累。对125I-AII仅与RIE-1细胞上酸可进入位点的浓度依赖性结合进行Scatchard分析，结果显示与从总125I-AII结合（酸可进入和不可进入位点）获得的KD值差异不大，但位点数量仅约为15%。
