Flow cytometric analysis of internal calcium mobilization via a B2-bradykinin receptor on a subclone of PC-12 cells.
Single cell Ca2+ mobilization was studied by nonparametric, 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 microM) 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 microM) 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 microM) in a concentration-dependent manner. Des-Arg9-Leu8-BK, a B1-receptor antagonist, failed to block the BK responses at 0.1-10 microM. 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.
Ransom
J T
JT
Institute of Molecular Immunology, Syntex Research, Palo Alto, California 94304.
Cherwinski
H M
HM
Dunne
J F
JF
Sharif
N A
NA
D000310:Adrenal Gland Neoplasms / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D005434:Flow Cytometry; D010673:Pheochromocytoma / Q000378:metabolism* / Q000473:pathology; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D012146:Rest; D014407:Tumor Cells, Cultured
D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; D001920:Bradykinin; D002118:Calcium