ATP-induced Secretion in PC12 Cells and Photoaffinity Labeling of Receptors |
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Authors: | Allen R Rhoads Rabin Parui Ngoc-Diep Vu Robert Cadogan Paul D Wagner |
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Institution: | Department of Biochemistry and Molecular Biology, Howard University College of Medicine, Washington, D.C.;National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. |
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Abstract: | Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P2-type pur-inergic receptor. ATP-induced norepinephrine release was inhibited 80% when extracellular Ca2+ 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 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 -tetradecanoyl-phorbol 13-acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, α-32P]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 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. |
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Keywords: | Catecholamine a xtracellular AT C12 cell orepinephrine releas 2 purinergic receptor |
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