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Multiple ecto‐nucleotidases in PC12 cells: identification and cellular distribution after heterologous expression
Author(s) -
Vollmayer Petra,
Koch Marco,
Braun Norbert,
Heine Petra,
Servos Jörg,
Israr Eltahmash,
Kegel Birgit,
Zimmermann Herbert
Publication year - 2001
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2001.00480.x
Subject(s) - 5' nucleotidase , extracellular , biology , biochemistry , nucleotide , nucleoside triphosphate , alkaline phosphatase , nucleoside , nucleotidase , phosphatase , microbiology and biotechnology , enzyme , gene
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 µ m and 243 µ m , 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.