ATP secretion and extracellular protein phosphorylation by CNS neurons in primary culture

The significant role of secreted ATP in the regulation of neuronal function and the activity of ecto-protein kinases which utilize extracellular ATP to phosphorylate proteins localized at the cell surface have been previously studied in peripheral neurons and in cloned neural cell lines. In the present study we have utilized neostriatal neurons differentiated in primary culture to demonstrate vesicular secretion of ATP and phosphorylation of proteins by extracellular ATP in neurons derived from the central nervous system (CNS). Neostriatal neurons from embryonic mice were maintained in a chemically defined medium for 15-18 days. Functional differentiation was determined by measuring evoked GABA-release. ATP-secretion was measured by luciferin-luciferase assays, and protein phosphorylation by adding gamma-32P-ATP to the extracellular medium. Depolarization by 50 mM KCl induced a Ca++-dependent ATP release, and stimulation by 100 microM veratridine resulted in secretion of ATP that could be blocked by tetrodotoxin. Phosphorylation of specific protein components with apparent molecular mass of 110 Kd, 80 Kd, 55 Kd, 30 Kd and 20 Kd was detected in striatal neurons incubated for 15 min with gamma-32P-ATP added to the medium, but not by labeling intracellular ATP pools with equivalent amounts of radioactivity presented as inorganic 32Pi. These results open for investigation the role of extracellular protein phosphorylation systems in processes underlying the responsiveness of CNS neurons to secreted ATP.