[3H]Adenosine Transport in Synaptoneurosomes of Postmortem Human Brain

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Abstract
[3H]Adenosine transport was characterized in cerebral cortical synaptoneurosomes prepared from postmortem human brain using an inhibitor-stop/centrifugation method. The adenosine transport inhibitors dipyridamole and dilazep completely and rapidly blocked transmembrane fluxes of [3H]adenosine. For 5-s incubations, two kinetically distinguishable processes were identified, i.e., a high-affinity adenosine transport system with Kt and Vmax values of 89 microM and 0.98 nmol/min/mg of protein, respectively, and a low-affinity adenosine transport system that did not appear to be saturable. For incubations with 1 microM [3H]adenosine as substrate, intrasynaptoneurosomal concentrations of [3H]adenosine were 0.26 microM at 5 s and 1 microM at 600 s. Metabolism of accumulated [3H]adenosine to adenine nucleotides was 15% for 5-s, 23% for 15-s, 34% for 30-s, 43% for 60-s, and 80% for 600-s incubations. The concentrations (microM) of total accumulated 3H-purines ([3H]adenosine plus metabolites) at these times were 0.3, 0.5, 1.0, 1.3 and 5.6, respectively. These results indicate that in the presence of extensive metabolism, the intrasynaptoneurosomal accumulation of 3H-purines was higher than the initial concentration of 1 microM [3H]adenosine in the reaction medium. For 5-, 15-, 30-, 60-, and 600-s incubations in the presence of the adenosine deaminase inhibitor EHNA and the adenosine kinase inhibitor 5'-iodotubercidin, metabolism of the transported [3H]adenosine was 14, 14, 16, 14, and 38%, respectively. During these times, total 3H-purine accumulation was 0.3, 0.5, 0.5, 0.7, and 1.8 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)