ATP-induced secretion in PC12 cells and photoaffinity labeling of receptors

Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P2-type purinergic receptor. ATP-induced norepinephrine release was inhibited 80% when extracellular Ca2+ was absent. Only four nucleotides, ATP, ATP gamma S, benzoylbenzoyl ATP (BzATP), and 2-methylthio-ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP-induced secretion was inhibited by Mg2+, and this inhibition was overcome by the addition of excess ATP suggesting that ATP4- 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-tetradecanoylphorbol 13-acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [alpha-32P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP gamma S and BzATP but not GTP blocked labeling of the proteins by [32P]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 P2 receptor responsible for ATP-stimulated secretion in these cells.