Effect of saponin and filipin on antagonist binding to AT1 receptors in intact cells

In the present study, [H-3]-candesartan binding experiments were performed on intact Chinese Hamster Ovary cells transfected with the human AT(1) receptor (CHO-AT(1) cells). Cells were pre-treated with 0.01 mg/ml saponin or filipin. Both pre-treatments resulted in all increased dissociation rate and decreased affinity of the insurmountable non-peptide antagonist [H-3]-candesartan. A similar decrease in affinity was observed for the peptide antagonist Sar(1)-Ile(8) angiotensin II and for other non-peptide antagonists, irrespectively of their degree of insurmountability. A similar discrepancy in [H-3]-candesartan binding was earlier observed when comparing intact CHO-AT(1) cells and membrane preparations thereof. This similarity is further highlighted by the observations that saponin or filipin no longer affect [H-3]-candesartan binding to CHO-AT(1) cell membranes and that both agents permeabilise the CHO-AT(1) cells. This suggests that the intracellular composition and/or organisation of living cells play an active role with regard to antagonist-AT(1) receptor interactions. (C) 2004 Elsevier Inc. All rights reserved.