USE OF SCANNING CYTOMETRY IN STUDYING BRADYKININ BINDING IN MRC-5 CELLS

Ligand-receptor affinity is classically demonstrated by measuring ligand binding density to a specific site on membrane preparations, and receptor function is studied by measuring calcium flux, cell by cell, using microspectrofluorimetry. In order to study these phenomena in a larger cell population, calcium flux was measured in MRC-5 cell line expressing the B-2 receptor for bradykinin using an ACAS 570 scanning cytometer. Following incorporation of fluo3/AM, different ligands were studied, singly or in association with bradykinin. This study confirmed that only the B-2 receptor is present on the plasma membrane of MRC-5 cells. Bradykinin binding to the B-2 receptor was not modified by a B-1 agonist (Des-Arg(9)-bradykinin) or by a B-1 antagonist (Des-Arg(9)-[Leu(8)]-bradykinin) but was inhibited by a B-2 agonist ([Hyp(3)]-bradykinin) and a B-2 antagonist (HOE 140). The source of free calcium was also studied in comparison with ionomycin. The intensity of the calcium peak after binding of bradykinin is independent of the concentration of extracellular calcium. Preincubation with diltiazem or TMB-8 did not modify calcium flux, indicating that transduction of the signal after bradykinin binding in this cell line is independent of voltage-dependent channels and does not require mobilization of intracellular calcium blocked by TMB-8. In conclusion, scanning cytometry can be used to study ligand-receptor binding and to obtain results rapidly from multiple cells. Recording of individual cell variations and kinetics enables identification of active agonists or antagonists and consequently the selection of new compounds.