Further characterization of an adenosine transport system in the mitochondrial fraction of rat testis

Previous work from our laboratory has demonstrated the presence of high-aff inity binding sites for [H-3]nitrobenzylthioinosine ([H-3]NBTI), a marker o f adenosine uptake systems, in the mitochondrial fraction of rat testis. He re, 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 pr otein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently i nhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC50 = 3 nM) although N BTI inhibited this uptake weakly (IC50 = 72.7 +/- 37.1 mu M). Dilazep > dip yridamole greater than or equal to hexobendine inhibited [H-3]adenosine inc orporation at low micromolar concentrations. The nucleosides inosine and ur idine were weak inhibitors of this system. The adenosine receptor ligands N -6-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptak e only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenos ine (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 carb oxamide (PK 11195) were able to inhibit it. The lack of inhibition by the b lockers 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 existen ce 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.