thioinosine and 2-hydroxy-5-nitrobenzylthioguanosine

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [3H]nitrobenzylthioinosine ([3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [3H]adenosine uptake assays. This system (K(m)=2+/-1.3 microM; 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, IC(50)=3 nM) although NBTI inhibited this uptake weakly (IC(50)=72. 7+/-37.1 microM). Dilazep>dipyridamole>/=hexobendine inhibited [3H]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 [3H]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.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Benzodiazepinones; Biological Transport; Dilazep; Dipyridamole; Dose-Response Relationship, Drug; Guanosine; Hexobendine; Hydrogen-Ion Concentration; Inosine; Isoquinolines; Kinetics; Male; Mitochondria; Osmolar Concentration; Phenylisopropyladenosine; Rats; Rats, Sprague-Dawley; Sodium; Subcellular Fractions; Temperature; Testis; Thioinosine; Thionucleosides; Time Factors; Tritium; Uridine

Previous studies indicated that the peptide neurotensin (NT) stimulates Cl(-) secretion in animal small intestinal mucosa in vitro. In this study, we investigated whether NT causes Cl(-) secretion in human colonic mucosa and examined the mechanism of this response.. Human mucosal preparations mounted in Ussing chambers were exposed to NT. Drugs for pharmacologic characterization of NT-induced responses were applied 30 minutes before NT.. Serosal, but not luminal, administration of NT (10(-8) to 10(-6) mol/L) induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive short-circuit current (Isc) increase that was inhibited by the specific nonpeptide NT receptor antagonists SR 48692 and SR 142948A, the neuronal blocker tetrodotoxin, and the prostaglandin synthesis inhibitor indomethacin. The mast cell stabilizer lodoxamide and the histamine 1 and 2 receptor antagonists pyrilamine and ranitidine, respectively, did not significantly alter NT-induced Isc increase. In contrast, the adenosine receptor 1 and 2 antagonists inhibited this secretory response, whereas the adenosine uptake inhibitors S-(4-nitrobenzyl)-6-thioguanosine and S-(4-nitrobenzyl)-6-thioinosine and the adenosine deaminase inhibitor deoxycoformycin potentiated NT-induced Isc increase. Serosal adenosine induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive Isc increase.. NT stimulates chloride secretion in human colon by a pathway(s) involving mucosal nerves, adenosine, and prostaglandins.

Topics: Adamantane; Adenosine; Adenosine Deaminase Inhibitors; Affinity Labels; Anti-Inflammatory Agents, Non-Steroidal; Cells, Cultured; Chlorides; Colon; Electrophysiology; Enteric Nervous System; Enzyme Inhibitors; Guanosine; Histamine; Humans; Imidazoles; In Vitro Techniques; Indomethacin; Intestinal Mucosa; Mast Cells; Membrane Potentials; Neurotensin; Pentostatin; Pyrazoles; Quinolines; Receptors, Neurotensin; Tetrodotoxin; Thioinosine; Thionucleosides

Site-specific binding to human erythrocyte membranes of nitrobenzylthioinosine (NBMPR), un-ionized at physiological pH, was compared with that of hydroxynitrobenzylthioinosine (HNBMPR), pKa 6.4, at graded pH values. Binding of [3H]NBMPR was measured directly, and that of HNBMPR was assayed by competitive inhibition by HNBMPR of [3H]NBMPR binding. Kd and Bmax values for binding of [3H]NBMPR to erythrocyte membranes were independent of pH. Kd values for the competing ligand were determined by mass law analysis of equilibrium binding data using either (a) apparent ligand concentration (dissociated plus undissociated forms of HNBMPR) or (b) the concentration of undissociated HNBMPR. Kd values for HNBMPR calculated with the apparent ligand concentration increased 10-fold as the fraction of HNBMPR molecules present in the dissociated form was increased (by pH changes) from 14 to 88%, whereas Kd values for the undissociated form of HNBMPR were independent of pH. The results presented here demonstrate that the undissociated form of HNBMPR binds more tightly to the transport-inhibitory sites of erythrocytes than NBMPR and suggest that ionization of S6-substituted thiopurine ribonucleosides eliminates or greatly decreases their ability to interact with the binding sites.

Topics: Affinity Labels; Binding, Competitive; Blood Proteins; Carrier Proteins; Erythrocyte Membrane; Guanosine; Humans; Hydrogen-Ion Concentration; Inosine; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Thioinosine; Thionucleosides

A major action of adenosine is the induction of profound behavioral inactivity. 6-(4-Nitrobenzyl)-thioinosine (NBI) and 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine (NBG) have been shown to inhibit adenosine uptake. To investigate the possible synergism between exogenous ligand and reuptake inhibition, mice were treated with NBI or NBG + adenosine. NBI and NBG potentiated the effects of adenosine at doses which did not in themselves induce behavioral inactivity. These behavioral results support the proposed role of NBI and NBG as adenosine uptake site blockers which increase synaptic concentrations of adenosine and postsynaptic responses to adenosine in vivo.

Topics: Adenosine; Animals; Arousal; Brain; Guanosine; Inosine; Male; Mice; Mice, Inbred C57BL; Motor Activity; Receptors, Cell Surface; Receptors, Purinergic; Synapses; Thioinosine; Thionucleosides