Transport and Metabolism of D‐[3H]Adenosine and L‐[3H]Adenosine in Rat Cerebral Cortical Synaptoneurosomes

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Abstract
The relationship between transport and metabolism in synaptoneurosomes was examined to determine the metabolic stability of rapidly accumulated D‐[3H]adenosine and L‐[3H]adenosine and the degree to which metabolism of the accumulated purines affected measurements of apparent KT and Vmax values for adenosine transport. For D‐[3H]adenosine, high‐ and low‐affinity accumulation processes were present. For the high‐affinity system an inverse relationship was found between transport reaction times and KT and Vmax values. For incubations of 5, 15, and 600 s, which corresponded to 24, 32, and 76% phosphorylation of accumulated D‐[3H]adenosine to nucleotides, apparent KT values were 9.4, 8.4, and 4.5 μM, respectively, and Vmax values were 850, 70, and 12 pmol/min/mg of protein, respectively. Pretreatment with 10 μM erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine, an adenosine deaminase inhibitor, and 5′‐iodotubercidin, an adenosine kinase inhibitor, decreased the phosphorylation of accumulated D‐[3H]adenosine to 6% with 5‐s and 9% with 15‐s incubations. This resulted in significantly higher KT values: 36 μM at 5 s and 44 μM at 5 s. At 10‐min incubations in the presence of these inhibitors, metabolism of accumulated D‐[3H]adenosine was 32%, and apparent KT and Vmax values at this time were not significantly different from those obtained without inhibitors. For L‐[3H]adenosine, apparent KT and Vmax values for 20‐s incubations were 38.7 μM and 330 pmol/min/mg of protein, respectively. Metabolism (mainly phosphorylation) of accumulated L‐[3H]adenosine was observed only at incubations of >30 s. Taken together, these results demonstrate that adenosine transport is significantly faster than subsequent metabolism; that accumulated D‐adenosine is rapidly incorporated into and trapped intracellularly as adenine nucleotides, thereby affecting measured kinetic parameters for adenosine transport and giving an “appearance” of concentrative accumulations; and that the apparent KTT value of 39 μM for D‐adenosine transport conducted in the presence of the enzyme inhibitors was the same as the apparent KT value for L‐adenosine transport.