Abstract

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [H-3]nitrobenzylthioinosine ([H-3]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [H-3]adenosine uptake assays. This system (K-m = 2 +/- 1.3 mu M; V-max = 86.2 +/- 15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC50 = 3 nM) although NBTI inhibited this uptake weakly (IC50 = 72.7 +/- 37.1 mu M). Dilazep > dipyridamole greater than or equal to hexobendine inhibited [H-3]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N-6-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4'-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and alpha,beta-methylene-ATP, indicates that [H-3]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm. (C) 2000 Elsevier Science B.V. All rights reserved.