Effects of peptidase inhibitors on binding at angiotensin receptor subtypes in the rat brain

Sulfhydryl reducing agents affect angiotensin II (AII) receptor binding differentially at AT1 and AT2 sites. 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 AT1 and AT2 binding sites, whereas bacitracin (10(-4) M) did not affect binding at either site. In contrast, addition of phenanthroline and bovine serum albumin (BSA) increased binding at AT1 sites 2.3-fold, whereas binding at AT2 sites was affected minimally. Degradation of 125I-[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 of 125I-SIAII is minimal in large volume in vitro receptor 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.