Multiple ecto-nucleotidases in PC12 cells: identification and cellular distribution after heterologous expression.
The physiological action of extracellular ATP and other nucleotides in the nervous system is controlled by surface-located enzymes (ecto-nucleotidases) of which several families with partially overlapping substrate specificities exist. In order to identify ecto-nucleotidases potentially associated with neural cells, we chose PC12 cells for analysis. PC12 cells revealed surface-located ATPase and ADPase activity with apparent K(m)-values of 283 microM and 243 microM, respectively. Using PCR we identified the mRNA of all members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated (NTPDase1 to NTPDase3, NTPDase5/6), of ecto-nucleotide pyrophosphatase/phosphodiesterase3 (NPP3), tissue-non-specific alkaline phosphatase and ecto-5'-nucleotidase. The surface-located catalytic activity differed greatly between the various enzyme species. Our data suggest that hydrolysis of ATP and ADP is mainly due to members of the ecto-nucleoside triphosphate diphosphohydrolase family. Activity of ecto-5'-nucleotidase and alkaline phosphatase was very low and activity of NPP3 was absent. For a detailed analysis of the cellular distribution of ecto-nucleotidases single and double transfections of PC12 cells were performed, followed by fluorescence analysis. Ecto-nucleotidases were distributed over the entire cell surface and accumulated intracellularly in varicosities and neurite tips. PC12 cell ecto-nucleotidases are likely to play an important role in terminating autocrine functions of released nucleotides and in producing extracellular nucleosides supporting the survival and neuritic differentiation of PC12 cells.
Vollmayer
P
P
Biozentrum der J.W. Goethe-Universität, AK Neurochemie, Zoologisches Institut, Frankfurt am Main, Germany.
Koch
M
M
Braun
N
N
Heine
P
P
Servos
J
J
Israr
E
E
Kegel
B
B
Zimmermann
H
H
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002454:Cell Differentiation / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D006224:Cricetinae; D004789:Enzyme Activation / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D008565:Membrane Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D016716:PC12 Cells; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D014162:Transfection
D008565:Membrane Proteins; D012333:RNA, Messenger; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases; C027201:ectoATPase