thioinosine and propentofylline

Adenosine receptor signal transduction mechanisms have previously been characterized in Syrian hamster smooth muscle DDT1 MF-2 cells but adenosine transport in these cells has not. DDT1 MF-2 cells possess a high density (370,000 sites/cell) of high affinity (Kd value of 0.26 nM) binding sites for [3H]nitrobenzylthioinosine, a marker for the equilibrative and inhibitor-sensitive subtype of nucleoside transporters. Transport of [3H]adenosine was insensitive to Na+ and was inhibited by the nucleoside transport inhibitors nitrobenzylthioinosine, dilazep and dipyridamole with IC50 values of 1, 13 and 270 nM, respectively. Propentofylline, a neuroprotective compound that can inhibit nucleoside transporters, is rapidly metabolized in vivo to the racemate (+/-)-A720287. Based on recent findings that some transport inhibitors exhibit marked stereoselectivity, we tested the degree to which individual stereoisomers of (+/-)-A720287 affect adenosine transport. Propentofylline inhibited [3H]adenosine transport in DDT1 MF-2 cells with an IC50 value of 24 microM. (+/-)-A720287 and the individual stereoisomers (+)-833791 and (-)-844261 had similar potency to propentofylline for inhibition of [3H]adenosine transport in DDT1 MF-2 cells as well as in clonal mouse leukemia L1210/B23.1 cells, cells which possess only the equilibrative and inhibitor-sensitive subtype of nucleoside transporters. Thus, the neuroprotective effects of propentofylline may be due, in part, to the primary metabolites of propentofylline.

Topics: Adenosine; Animals; Binding Sites; Biological Transport; Cricetinae; Dilazep; Dipyridamole; Genital Neoplasms, Male; Kinetics; Leiomyosarcoma; Leukemia L1210; Male; Mesocricetus; Muscle, Smooth; Rats; Thioinosine; Tumor Cells, Cultured; Vas Deferens; Xanthines

The neuroprotective effects of adenosine are well-recognized. Recently, propentofylline, a xanthine derivative, has been shown to increase extracellular concentrations of adenosine in ischemic brain and to limit the extent of neuronal damage in experimental models of cerebral ischemia. Since the concentration of adenosine in brain is controlled, in part, by nucleoside transporter proteins, the action of propentofylline was proposed to be due to inhibition of mediated transfer of adenosine across cell membranes. To determine the likelihood of this mechanism, we examined the inhibitory effects of propentofylline on [3H]adenosine transport by the three best-characterized nucleoside transport processes, es, ei, and cif in cultured cell lines under conditions where only a single transporter type was operative. Propentofylline inhibited [3H]adenosine uptake by each of the three transport processes in a concentration-dependent manner. The greatest inhibitory potency was for es transporters (L1210/B23.1 cells), with an IC50 value of 9 microM, followed by ei transporters, with IC50 values of 170 microM (L1210/C2 cells) and 166 microM (Walker 256 cells). Propentofylline was a weak inhibitor of cif transporter, with an IC50 value of 6 mM. These results demonstrate that propentofylline is an inhibitor of adenosine transport processes and suggest that its neuroprotective effects may be due to an increase in extracellular concentrations of adenosine by virtue of inhibition of es transporter function.

Topics: Adenosine; Animals; Biological Transport; Brain Ischemia; Cell Line; Mice; Rats; Thioinosine; Tritium; Tumor Cells, Cultured; Xanthines

Previous studies have demonstrated that the xanthine compound, propentofylline, has beneficial effects in models of cerebral ischemia and can enhance some and exhibit other effects of adenosine. We investigated the in vitro effects of propentofylline and its hydroxy metabolite, A72,0287, on the binding of [3H]cyclohexyladenosine ([3H]CHA), [3H]2-[p-(2-carbonyl-ethyl)-phenylethyl-amino]-5'-N- ethylcarboxamido adenosine ([3H]CGS 21680) and [3H]nitrobenzylthioinosine ([3H]NBMPR) to adenosine A1 and A2 receptors and NBMPR-sensitive nucleoside transporters, respectively, in 10-microns coronal rat brain sections. Both xanthines had micromolar affinity for each of these sites with approximately 10-fold lower affinity for A2 receptors than for A1 receptors and [3H]NBMPR binding sites. Saturation analysis of [3H]CHA or [3H]CGS 21680 binding in the presence of increasing concentrations of propentofylline produced significant increases in KD values without affecting Bmax values; thus propentofylline is a competitive inhibitor at A1 and A2 receptors. The effects on A2 receptors apparently require higher concentrations (Ki approximately 200 microM) than the effects on A1 receptors (Ki approximately 20 microM). Propentofylline was also found to be a competitive inhibitor of [3H]NBMPR binding. Therefore we conclude that propentofylline interacts with adenosine-responsive systems to increase interstitial adenosine concentrations and to selectively inhibit A1 receptors.

Topics: Adenosine; Animals; Autoradiography; Binding Sites; Brain; Carrier Proteins; Male; Membrane Proteins; Nucleoside Transport Proteins; Rats; Rats, Inbred Strains; Receptors, Purinergic; Thioinosine; Xanthines

Hippocampal cells were cultured in 24-well culture plates with enriched populations of neuron or glial cells. The [3H]adenosine uptake by 7-10-day cultures of these cells was dependent on temperature, but independent of extracellular Na+. The uptake of adenosine (10 microM) for 15 s was greatly blocked by addition of 100 microM dipyridamole, 50-200 microM propentofylline or 50 microM of 2-chloroadenosine or nitrobenzylthioinosine in both cells and by 100 microM pentoxifylline in neuron. Either caffeine or theophylline (50 microM each) had no effect on the uptake by these cells. Inhibition of the adenosine uptake by propentofylline was demonstrated to be competitive in both cells.

Topics: 2-Chloroadenosine; Adenosine; Affinity Labels; Animals; Biological Transport; Caffeine; Cells, Cultured; Cytarabine; Dipyridamole; Embryo, Mammalian; Hippocampus; Kinetics; Neuroglia; Neurons; Pentoxifylline; Rats; Sodium; Theophylline; Thioinosine; Tritium; Xanthines