title: Characterization of the B2 receptor and activity of bradykinin analogs in SHP-77 cell line by Cytosensor microphysiometer.
abstract: The Cytosensor microphysiometer device (Molecular Devices, Sunnyvale, CA) is capable of measuring the rate at which cells acidify their environment in response to ligand-receptor binding. By measuring the extracellular acidification response (ECAR) we characterized some aspects of ligand-B2 receptor interaction in SHP-77 cell line. SHP-77 cells maximally acidified their environment within 30 s after the exposure to bradykinin (BK) or the BK agonist, B9972, with the maximum effect seen at a ligands concentration of 1 microM. Fetal bovine serum (FBS) modulated the binding of BK or B9972, showing that B9972 is a partial agonist. In addition, the binding of BK agonist or antagonist to the B2 receptor showed different ECAR and different interaction with other intracellular and plasma membrane proteins. Our microphysiometrical results showed that two parameters, antagonist binding affinity (pD2) and antagonist potency (pIC50) are required to characterize BK antagonist activity for the B2 receptor in the SHP-77 cell line. The previously used parameter of B2 antagonist activity, pA2, had high variation and poor correlation with the inhibition of SHP-77 cell growth in vitro and suppression of tumor growth when SHP-77 cells were injected to mice. Our results permit us to conclude that BK agonists and antagonists differ in their interactions with the B2 receptor and consequently elicit different cell responses. Based on our results, we have developed a new microphysiometrical assay for analyzing the activity of BK agonists and antagonist in SHP-77 cells, which may facilitate the discovery of new potent anticancer drugs.
authors.affiliation: Department of Developmental Biology, Institute of Biochemistry, 2600 Vilnius, Lithuania. daiva@bchi.lt
authors.forename: Daiva
authors.initials: D
authors.lastname: Bironaite
authors.forename: Lajos
authors.initials: L
authors.lastname: Gera
authors.forename: John M
authors.initials: JM
authors.lastname: Stewart
mesh_terms: D000595:Amino Acid Sequence; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D065094:Bradykinin B2 Receptor Antagonists; D018288:Carcinoma, Small Cell / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008175:Lung Neoplasms / Q000378:metabolism*; D046208:Microchip Analytical Procedures; D011485:Protein Binding; D043782:Receptor, Bradykinin B2 / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism
chemical_list: D065094:Bradykinin B2 Receptor Antagonists; D043782:Receptor, Bradykinin B2; D017923:Sodium-Hydrogen Exchangers; D001920:Bradykinin