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 specificit ies exist. In order to identify ecto-nucleotidases potentially associated w ith neural cells, we chose PC12 cells for analysis. PC12 cells revealed sur face-located ATPase and ADPase activity with apparent K-m-values of 283 muM and 243 muM, 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/pho sphodiesterase3 (NPP3), tissue-non-specific alkaline phosphatase and ecto-5 ' -nucleotidase. The surface-located catalytic activity differed greatly b etween the various enzyme species. Our data suggest that hydrolysis of ATP and ADP is mainly due to members of the ecto-nucleoside triphosphate diphos phohydrolase family. Activity of ecto-5 ' -nucleotidase and alkaline phosph atase was very low and activity of NPP3 was absent. For a detailed analysis of the cellular distribution of ectonucleotidases single and double transf ections of PC12 cells were performed, followed by fluorescence analysis. Ec tonucleotidases were distributed over the entire cell surface and accumulat ed intracellularly in varicosities and neurite tips. PC12 cell ecto-nucleot idases are likely to play an important role in terminating autocrine functi ons of released nucleotides and in producing extracellular nucleosides supp orting the survival and neuritic differentiation of PC12 cells.