Detection of angiotensin II type 1 receptor ligands by a cell-based assay

Abstract

This work describes a cell-based assay that does not depend on radioactivity or laboratory animals for the detection of ligands of angiotensin II type 1 receptor (AT1R). The assay makes use of stable transfected Chinese hamster ovary cells (CHO-AT1R) expressing the AT1R. A sequential saturation assay principle was used in which receptor binding sites of the CHO-AT1R cells are blocked by the analyte in a concentration-dependent manner. Afterwards, TAMRA-angiotensin II, a fluorescence-labeled ligand, was added to bind to the remaining free binding sites of the receptor. In consequence, the fluorescence signal determined is inversely proportional to the concentration of the analyte.

Long‐lasting target binding and rebinding as mechanisms to prolong in vivo drug action

“…Intact plated cells seem to offer a compromise between these two in vitro experimental systems (Figure 6). From the physiological point of view, they certainly offer a more relevant environment than the membrane preparations, not only because they are alive but also because, to some extent, they mimic the microanatomic complexity of intact tissues (Spivak et al ., 2006; Grießner et al ., 2009). Yet, compared with those tissues, they offer far greater experimental flexibility, including the ability to compare radioligand binding and functional data under the same experimental conditions, easy wash‐out and the ability to parallel determinations on equivalent samples in each well rather than consecutive determinations on each tissue (Vauquelin et al ., 2002).…”

Cell membranes… and how long drugs may exert beneficial pharmacological activity in vivo

“…As they mimic the complexity of intact tissues, confluent cell monolayers represent convenient surrogate systems for the study of drug rebinding . Using recombinant Chinese hamster ovary (CHO) cells, we compared the dissociation profiles of different radioligand/receptor combinations in washout experiments in the presence and absence of an excess of unlabelled competitor to estimate the prominence of rebinding (simulated examples are shown in Figure A and B).…”

“…Moreover, the extracellular matrix as well as an unstirred layer of water molecules very close to the membrane constitute further obstacles that may hinder the approach and escape of drugs [83]. A comparable situation also occurs within the cell for drugs that cross the surrounding plasma membrane with some difficulty [7,74,84].As they mimic the complexity of intact tissues, confluent cell monolayers represent convenient surrogate systems for the study of drug rebinding [85][86][87]. Using recombinant Chinese hamster ovary (CHO) cells, we compared the dissociation profiles of different radioligand/receptor combinations in washout experiments in the presence and absence of an excess of unlabelled competitor to estimate the prominence of rebinding [74] (simulated examples are shown in Figure 4A and B).…”

Clozapine, atypical antipsychotics, and the benefits of fast-off D2 dopamine receptor antagonism

“…Quite similar distinctions have also been reported for GPCRs (Fierens et al 2002;Hara et al 1998;Smith et al 2006), and it is most striking that leaky cells already had the faculty to behave membrane-like (Verheijen et al 2004;Vauquelin and Packeu 2009). Finally, compared with membranes, plated cells mimic much better the microanatomic complexity of tissues (Spivak et al 2006;Grießner et al 2009;Vauquelin and Charlton 2010). Indeed, those cells are separated by clefts that somewhat mimic neuronal synapses and other interstitial spaces whose walls and macromolecular content hinders the free diffusion of ligands to and from their receptors (Spivak et al 2006;Cragg and Rice 2004;Hrabctová and Nicholson 2004).…”