A. Jimenez et al., Further characterization of an adenosine transport system in the mitochondrial fraction of rat testis, EUR J PHARM, 398(1), 2000, pp. 31-39
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.