Anales RANF

P.25 ECTO-NUCLEOTIDE PYROPHOSPHATASE/PHOSPHODIESTERASE (E-NPP) ACTIVITY IN NEURO-2A NEUROBLASTOMA CELLS J. Gualix, R. Gómez-Villafuertes, F. Ortega, M.T. Miras-Portugal School of Veterinary Sciences, Complutense University of Madrid (UCM), Madrid, Spain; Neurochemistry Research Institute, IUIN, UCM, Madrid, Spain; Sanitary Research Institute of the San Carlos Clinical Hospital, IdiSSC, UCM, Madrid, Spain. Diadenosine polyphosphates (Ap n As) comprise a group of compounds formed by two adenosine moieties linked by a phosphate chain of variable length. Ap n As fulfill with the requirements to be considered as signaling molecules in the central nervous system: they are co-stored with ATP and other neurotransmitters in storage granules of neural and neuroendocrine cells. The exocytotic release of these compounds permits them to interact with P2 receptors, both metabotropic and ionotropic. Moreover, Ap n As can also activate specific receptors termed dinucleotide receptors. Extracellular actions of Ap n As are finished by ectonucleotidases that degrade these compounds yielding adenosine as the final product. N2a cells display an ectoenzymatic hydrolytic activity able to degrade diadenosine polyphosphates. The Ap n A-cleaving activity of these cells has been analyzed with the use of the fluorogenic compound BODIPY-FL-GTP ɣS. Hydrolysis of this dinucleotide analog showed a hyperbolic kinetic with a K m value of 4.9 ± 1.3 μM. Ap 5 A, Ap 4 A, Ap 3 A as well as the nucleoside monophosphate AMP behaved as inhibitors of BODIPY-FL-GTP ɣS extracellular degrada tion. Ectoenzymatic activity shared the typical characteristics of the E-NPP family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of divalent cations, being strongly inhibited by EDTA and activated by Zn 2+ ions. Both NPP1 and NPP3 isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal-like phenotype. It is relevant to point the expression pattern of the NPP3 protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main isozyme involved in the extracellular degradation of dinucleotides in these cells, this enzyme reducing its activity and changing its subcellular location following neuronal differentiation.