ADENOSINE-INDUCED APOPTOSIS IN CHICK EMBRYONIC SYMPATHETIC NEURONS - A NEW PHYSIOLOGICAL-ROLE FOR ADENOSINE

1. A newly found action of adenosine in neurons, which may have an imp ortant physiological function in the growth and development of the sym pathetic nervous system, is described. Adenosine (1-100 mu M) inhibite d neurite outgrowth within the first 24 h and killed about 80% of symp athetic neurons supported by nerve growth factor over the next 2 days in culture. Neurons supported by excess KCl, forskolin or phorbol 12,1 3-dibutyrate were equally susceptible to the toxic actions of adenosin e. Inosine, guanosine or hypoxanthine (all 100-300 mu M) were without effect on neuronal growth and survival. 2. Specific agonists of adenos ine A(1) and A(2) receptors were not neurotoxic, and toxic effects of adenosine were not antagonized by aminophgylline. These results rule o ut involvement of adenosine receptors and the adenylyl cyclase-cAMP si gnalling system in neurotoxic actions of adenosine. 3. Adenosine toxic ity was prevented by inhibitors of the adenosine membrane transporter, suggesting an intracellular site of action of adenosine. 4. Inhibitor s of adenosine deaminase dramatically facilitated the toxic action so that physiologically relevant concentrations of adenosine were neuroto xic.5. Adenosine kinase activity of sympathetic neurons was dose-depen dently inhibited by 5'-iodotubercidin (3-100 nM). 5'-Iodotubercidin (1 00 nM) completely protected neurons against toxicity of adenosine plus adenosine deaminase inhibitors. These results provide convincing evid ence that phosphorylation of the nucleoside is an essential requiremen t for initiation of adenosine toxicity. 6. Sympathetic neurons were su ccessfully rescued from the lethal effects of adenosine deaminase inhi bitor plus adenosine by uridine or 2-deoxycytidine, but not by nicotin amide or 2-deoxyguanosine, suggesting that depletion of pyrimidine nuc leotides by phosphorylated adenosine compounds and consequent inhibiti on of DNA synthesis produces neuronal death. 7. DNA fragmentation, ass essed by the fluorescent dye bisbenzimide and by the TUNEL (terminal d eoxynucleotidyl transferase-mediated nick end labelling) method, indic ated that neuronal death induced by adenosine was apoptotic. 8. We con clude that adenosine deaminase and adenosine kinase play an important role in the metabolism of intracellular concentrations of adenosine an d thereby regulate the growth and development of sympathetic neurons. Our study highlights, for the first time, the importance of adenosine as a mediator of programmed cell death of neurons supported by nerve g rowth factor.