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Flow Cytometric Analysis of Internal Calcium Mobilization via a B 2 ‐Bradykinin Receptor on a Subclone of PC‐12 Cells
Author(s) -
Ransom J. T.,
Cherwinski H. M.,
Dunne J. F.,
Sharif N. A.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb02018.x
Subject(s) - receptor , receptor antagonist , bradykinin , population , microbiology and biotechnology , flow cytometry , agonist , chemistry , endocrinology , medicine , antagonist , biology , biochemistry , environmental health
Single cell Ca 2+ 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 Ca 2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK 1 , BK or the B 2 ‐BK receptor agonists Lys‐BK and Met‐Lys‐BK (10 p M ‐1 μ M ) induced robust Ca 2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B 1 ‐BK receptor agonist Des‐Arg 9 ‐BK (1 n M ‐1 μ M ) failed to elicit Ca 2+ mobilization in these cells. The responses to BK (10 and 100 n M ) were inhibited by preincubation with the B 2 ‐receptor antagonists D‐Arg 0 ‐Hyp 3 ‐thienyl 5,8 ‐D‐Phe 7 ‐BK and D‐Arg 0 ‐Hyp 3 ‐D‐Phe 7 (0.1 n M ‐10 μ M ) in a concentration‐dependent manner. Des‐Arg 9 ‐Leu 8 ‐BK, a B 1 ‐receptor antagonist, failed to block the BK responses at 0.1–10 μ M . The agonist/antagonist profile of the BK responses indicated that the B 2 ‐BK receptor mediated the Ca 2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor‐mediated Ca 2+ 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.