IDENTIFICATION OF RECEPTOR LIGANDS AND RECEPTOR SUBTYPES USING ANTAGONISTS IN A CAPILLARY ELECTROPHORESIS SINGLE-CELL BIOSENSOR SEPARATION SYSTEM

A capillary electrophoresis system with single-cell biosensors as a de tector has been used to separate and identify ligands in complex biolo gical samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions, The single-cell biosensor was b ased on the ligand-receptor binding and G-protein-mediated signal tran sduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion i ndicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradyk inin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HO E 140), a selective antagonist to the BK B-2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Struc turally related forms of BK were also identified based on inhibiting B 2BK receptors. Applications of this technique include identification o f endogenous BK in a lysate of human hepatocellular carcinoma cells (H ep G2) and screening for bioactivity of BK degradation products in hum an blood plasma, The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of sepa rated components and receptor subtypes.