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.
Rhoads
A R
AR
Department of Biochemistry and Molecular Biology, Howard University College of Medicine, Washington, D.C. 20059.
Parui
R
R
Vu
N D
ND
Cadogan
R
R
Wagner
P D
PD
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D000345:Affinity Labels; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism*; D016716:PC12 Cells; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D000345:Affinity Labels; D000470:Alkaloids; D018047:Receptors, Purinergic P1; D012265:Ribonucleotides; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D019311:Staurosporine; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D009638:Norepinephrine