Flow Cytometric Analysis of Internal Calcium Mobilization via a B2‐Bradykinin Receptor on a Subclone of PC‐12 Cells

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Abstract
Single cell Ca2+ mobilization was studied by non‐parametric, quantitative flow cytometry using a sort‐selected subclone of PC‐12 cells. The response of the parent PC‐12 population to bradykinin (BK) was very heterogeneous and of a relatively low magnitude. Cells that exhibited maximal Ca2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK1, BK or the B2‐BK receptor agonists Lys‐BK and Met‐Lys‐BK (10 pM‐1 μM) induced robust Ca2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B1‐BK receptor agonist Des‐Arg9‐BK (1 nM‐1 μM) failed to elicit Ca2+ mobilization in these cells. The responses to BK (10 and 100 nM) were inhibited by preincubation with the B2‐receptor antagonists D‐Arg0‐Hyp3‐thienyl5,8‐D‐Phe7‐BK and D‐Arg0‐Hyp3‐D‐Phe7 (0.1 nM‐10 μM) in a concentration‐dependent manner. Des‐Arg9‐Leu8‐BK, a B1‐receptor antagonist, failed to block the BK responses at 0.1–10 μM. The agonist/antagonist profile of the BK responses indicated that the B2‐BK receptor mediated the Ca2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor‐mediated Ca2+ response can be employed to select a homogeneously responsive subclone from a heterogeneous, clonal population that can improve the resolution of receptor‐mediated second messenger generation at the single cell level.