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ATP‐induced Secretion in PC12 Cells and Photoaffinity Labeling of Receptors
Author(s) -
Rhoads Allen R.,
Parui Rabin,
Vu NgocDiep,
Cadogan Robert,
Wagner Paul D.
Publication year - 1993
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.1471-4159.1993.tb09800.x
Subject(s) - photoaffinity labeling , secretion , receptor , chemistry , affinity label , biophysics , microbiology and biotechnology , neuroscience , pharmacology , biochemistry , biology
— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P 2 ‐type pur‐inergic receptor. ATP‐induced norepinephrine release was inhibited 80% when extracellular Ca 2+ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2‐methylthio‐ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP‐induced secretion was inhibited by Mg 2+ , and this inhibition was overcome by the addition of excess ATP suggesting that ATP 4‐ was the active ligand. ATP‐induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12‐ O ‐tetradecanoylphorbol 13‐acetate. The stimulatory effects of 12‐ O ‐tetradecanoyl‐phorbol 13‐acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α‐ 32 P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP‐γS and BzATP but not GTP blocked labeling of the proteins by [ 32 P]BzATP. Labeling of the 50‐kDa protein was more sensitive to competition by 2‐methylthio‐ATP than the other labeled proteins, suggesting that the 50‐kDa protein represents the P 2 receptor responsible for ATP‐stimulated secretion in these cells.