Production of Adenosine from Extracellular ATP at the Striatal Cholinergic Synapse

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Abstract
The components of the ectonucleotidase pathway at the immunoaffinity-purified striatal cholinergic synapse have been studied. The ecto-ATPase (EC 3.6.1.15) had a Km of 131 γM, whereas the ecto-ADPase (EC 3.6.1.6) had a Km of 58 γM, was Ca2+-dependent, and was inhibited by the ATP analogue 5′-adenylylimidodiphosphate (AMPPNP). The ecto-5′-nucleotidase (EC 3.1.3.5) had a Km of 21 γM, was inhibited by AMPPNP and α,β-methylene ADP, and by a specific antiserum. The Vmax values of the ATPase, ADPase, and 5′-nucleotidase enzymes present at this synapse were in a ratio of 30:14:1. Very little ecto-adenylate kinase activity was detected on these purified synapses. The intraterminal 5′-nucleotidase enzyme, which amounted to 40% of the total 5′-nucleotidase activity, was inhibited by AMPPNP, α,β-methylene ADP, and the antiserum, and also had the same kinetic properties as the ectoenzyme. The time course of ATP degradation to adenosine outside the nerve terminals showed a delay, followed by a period of sustained adenosine production. The delay in adenosine production was proportional to the initial ATP concentration, was a consequence of feedforward inhibition of the ADPase and 5′-nucleotidase, and was inversely proportional to the ecto-5′-nucleotidase activity. The function and characteristics of this pathway and the central role of 5′-nucleotidase in the regulation of extraterminal adenosine concentrations are discussed.