Effects of peptidase inhibitors on binding at angiotensin receptor subtypes in the rat brain.
Effects of peptidase inhibitors on binding at angiotensin receptor subtypes in the rat brain.
复制标题
肽酶抑制剂对大鼠脑血管紧张素受体亚型结合的影响。
DOI：
10.1016/0006-2952(93)90023-p
复制
发表时间：
1993
影响因子：
5.8
通讯作者：
Rowe,BP
中科院分区：
文献类型：
--
作者：
Saylor,DL;Speth,RC;Rowe,BP
巯基还原剂对血管紧张素II（AII）受体AT 1和AT 2位点结合的影响不同。因此，巯基还原剂现在很少用于AII受体结合试验。在这方面，目前的放射自显影研究评价的影响，其他肽酶抑制剂对AII受体结合和放射性配体的完整性。5 mM EDTA在AT 1和AT 2结合位点上的结合也类似地增强了约70%，而杆菌肽（10− 4 M）对两个位点的结合都没有影响。相比之下，此外菲咯啉和牛血清白蛋白（BSA）增加结合在at 1 sites 2.3倍，而结合在AT 2网站的影响最小。在每种缓冲液中与组织孵育前后，通过HPLC分析样品，测定125 I [Sar 1，Ile 8]-AII（125 I-SIAII）的降解。从缓冲液中省略杆菌肽将完整放射性配体的回收率降低至83- 87%，而在存在或不存在所有其他缓冲液组分的情况下，回收率超过94%。这些结果表明，在大鼠脑AII受体的大体积体外受体放射自显影研究中，125 I-SIAII的降解是最小的。此外，对放射性配体结合的有利影响所造成的缓冲液成分，如EDTA，菲咯啉，和BSA不是由于他们的能力，以保护放射性配体从酶降解。由于这些成分（可能还有其他成分）对受体亚型的结合有不同的影响，因此在解释或比较从使用不同缓冲液成分的各个实验室获得的结合数据时应谨慎。
Sulfhydryl reducing agents affect angiotensin II (AII) receptor binding differentially at AT1and AT2sites. Consequently, sulfhydryl reducing agents are now used infrequently in AII receptor binding assays. In this regard, the present autoradiographic study evaluates the effects of additional peptidase inhibitors on AII receptor binding and radioligand integrity. EDTA at 5 mM enhanced binding similarly, by about 70%, at both AT1and AT2binding sites, whereas bacitracin (10−4M) did not affect binding at either site. In contrast, addition of phenanthroline and bovine serum albumin (BSA) increased binding at at1sites 2.3-fold, whereas binding at AT2sites was affected minimally. Degradation of125I [Sar1,Ile8]-AII (125I-SIAII) was determined by HPLC analysis of samples before and after incubation with tissue in each buffer. Omission of bacitracin from buffers reduced the recovery of intact radioligand to 83–87%, while recovery exceeded 94% in the presence or absence of all other buffer constituents. These results suggest that degradation of125I-SIAII is minimal in large volumein vitroreceptor autoradiography studies of rat brain AII receptors. Further, the beneficial effects on radioligand binding caused by buffer constituents such as EDTA, phenanthroline, and BSA were not due to their ability to protect the radioligand from enzymatic degradation. Because these constituents (and possibly others) had differential effects on binding with respect to receptor subtypes, caution should be used when interpreting or comparing binding data obtained from various laboratories utilizing different buffer components.