Characterization of ATP receptor which mediates norepinephrine release in PC12 cells.
PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.
Majid
M A
MA
Department of Physical Biochemistry, Gunma University, Maebashi, Japan.
Okajima
F
F
Kondo
Y
Y
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D009638:Norepinephrine / Q000378:metabolism*; D016716:PC12 Cells / Q000187:drug effects* / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D011983:Receptors, Purinergic / Q000187:drug effects* / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology
D000345:Affinity Labels; D015220:Calcium Channels; D007295:Inositol Phosphates; D011983:Receptors, Purinergic; D014227:Triazines; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; C012842:Cibacron Blue F 3GA; D013498:Suramin; D011189:Potassium Chloride; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D001920:Bradykinin; D002118:Calcium; D009638:Norepinephrine