Inhibitory effect of nonsteroidal anti-inflammatory drugs on adenosine transport in vascular smooth muscle cells.

It is generally accepted that the clinical efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) arises mainly from the inhibition of cyclooxygenase (COX). However, more evidence has suggested that certain pharmacological actions of NSAIDs may be mediated by COX-independent mechanisms. The present study investigated the effects of NSAIDs on adenosine uptake in human aortic smooth muscle cells (HASMCs). Among the NSAIDs tested (all at 100 microM), aspirin, ibuprofen and naproxen had no effect on [(3)H]adenosine uptake. Piroxicam inhibited [(3)H]adenosine uptake by 30%, while etodolac, indomethacin, ketoprofen, mefenamic acid and sulindac inhibited [(3)H]adenosine by 13-18%. Sulindac sulfide, an active metabolite of sulindac, inhibited [(3)H]adenosine uptake and [(3)H]nitrobenzylmercaptopurine ribonucleoside (NBMPR) binding of HASMCs with IC(50) values of 40.67+/-4.82 and 24.19+/-3.76 muM, respectively. Kinetic studies revealed that sulindac sulfide was a competitive inhibitor of adenosine uptake. Using the nucleoside-transporter-deficient PK15NTD cells that stably express equilibrative nucleoside transport (ENT) 1 and ENT2, it was found that the inhibitory effect of sulindac sulfide on ENT1 was greater than that on ENT2. Sulindac sulfide increased the extracellular adenosine level. In addition, it inhibited the proliferation of HASMCs and this anti-proliferative effect could be abolished by adenosine A(2B) receptor antagonist. Our results suggest that sulindac sulfide may exert pharmacological effects through the inhibition of adenosine uptake, which modulates the availability of adenosine in the vicinity of adenosine receptors.

Sensitivity to inhibition by N-ethylmaleimide: a property of nitrobenzylthioinosine-sensitive equilibrative nucleoside transporter of murine myeloma cells.

Murine myeloma SP2/0-Ag14 cells possess both nitrobenzylthioinosine (NBMPR)-sensitive and NBMPR-insensitive equilibrative uridine transport systems. No Na(+)-dependent uridine transport system was detected. The NBMPR-insensitive transport system is similarly insensitive to inhibition by dilazep and dipyridamole. Dose-response curve for the inhibition of equilibrative uridine transport by N-ethylmaleimide (NEM), a sulfhydryl reagent, in these cells was biphasic. About 30-40% of the uridine transport was inhibited by NEM at IC50 value of 0.15 mM. The other 60-70% of the transport activity remained insensitive to NEM at concentration as high as 3 mM. The decrease in NBMPR-sensitive uridine transport in the presence of 0.3 mM NEM was due to a 3-fold decrease in transport affinity. Apparent Km values of 500 and 1600 microM and Vmax values of 13 and 12 microM/s were obtained for untreated and NEM-treated cells, respectively. NEM (0.3 mM) has little effect on the Km of NBMPR-insensitive transporter, with apparent Km values of 100 and 110 microM and Vmax values of 3.0 and 2.5 microM/s for untreated and NEM-treated cells, respectively. High sensitivity of NBMPR-sensitive transporter to NEM inhibition was also observed in HL-60 and MCF-7 cells. Decrease in specific 3H-NBMPR equilibrium binding affinity in myeloma cells was observed after treatment with 0.3 mM NEM. Apparent Kd values of 0.32 and 2.3 nM with Bmax values of 48,000 and 44,000 sites/cell were obtained for untreated and NEM-treated cells, respectively. NBMPR, dilazep and dipyridamole at 30 microM, and uridine at 10 mM failed to protect the NBMPR-sensitive transporter against NEM inhibition. It is possible that a critical sulfhydryl residue is closed to substrate binding/transporting site of the NBMPR-sensitive transporter. NEM, a sulfhydryl reagent containing an activated double bond, hinders the affinity of this transporter by forming a stable thiol ether bond with the reactive residue.

Enhanced functional preservation of cold-stored rat heart by a nucleoside transport inhibitor.

This study investigates the hypothesis that inhibition of nucleoside transport during hypothermic storage elevates tissue adenosine (ADO) content and improves the function of the isolated rat heart. The hearts, flushed with a cardioplegic solution containing varying concentrations (0-100 nM) of a nucleoside transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI), were immersion-stored at 0 degrees C for 9 hr. Function was assessed after 30 min of working reperfusion. Function of unstored fresh hearts served as controls and poststorage recovery is reported as percentage of control function. Poststorage heart rate in all groups returned to control level after reperfusion. Recovery of other functional parameters in the no-NBTI group was as follows: aortic flow (AF), 56.2 +/- 4.6%; coronary flow (CF), 53.9 +/- 3.2%; cardiac output (CO), 55.5 +/- 4.0%; systolic pressure, 81.6 +/- 2.5%; work, 47.0 +/- 4.2%; and coronary vascular resistance (CVR), 157.1 +/- 7.8% of control. NBTI improved functional recovery in a dose-dependent fashion; the maximal improvement was seen at a dose of 5 nM, in which the recovery was: AF, 78.1 +/- 3.4%; CF, 73.5 +/- 4.4%; CO, 76.7 +/- 3.6%; work, 70.7 +/- 5.0%; and CVR, 127.5 +/- 4.5% of control (P < 0.05 vs. no-NBTI). The ADO A1-receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (0.1 microM) blocked the effects of 5 nM NBTI; the recovery of AF, CF, CO, work, and CVR decreased to 62.8 +/- 8.0%, 58.3 +/- 5.0%, 61.5 +/- 3.9%, 54.4 +/- 4.5%, and 163.8 +/- 12.7% of control, respectively (P < 0.05 vs. 5 nM NBTI). Tissue ADO content in 5 nM NBTI hearts at the end of storage was 0.075 +/- 0.025 mumol/g dry wt, which was significantly elevated from 0.016 +/- 0.004 mumol/g dry wt in no-NBTI hearts. Purine release during initial reperfusion was delayed in 5 nM NBTI hearts, indicating the inhibition of nucleoside transport by NBTI. But NBTI treatment did not improve end-storage or end-reperfusion myocardial ATP. In conclusion, the addition of NBTI to cardioplegic solution enhanced tissue ADO and improved poststorage function of the hypothermically stored rat heart. The effect is ADO A1-receptor mediated without invoking energy conservation.

Inhibitory effects of propentofylline on [3H]adenosine influx. A study of three nucleoside transport systems.

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.

Facilitated diffusion and sodium-dependent transport of purine and pyrimidine nucleosides in rat liver.

In mammalian cells, nucleoside transport usually is mediated by facilitated diffusion. In addition, a Na(+)-dependent, concentrative nucleoside transport system has been detected in several tissues but not the liver. To further clarify hepatic nucleoside transport mechanisms, we measured the uptake of [2-14C]uridine (2 to 100 mumol/L) and of [8-14C]adenosine (10 to 75 mumol/L) by the isolated perfused rat liver in the presence or absence of extracellular sodium or specific inhibitors of facilitated nucleoside diffusion. Uridine transport and metabolism were monitored by the release of labeled catabolites including 14CO2, which indicated complete degradation of the pyrimidine. Adenosine, uridine and uridine catabolites were measured in the effluent perfusate by reversed-phase high-performance liquid chromatography and a radioactivity flow monitor. The existence of a Na(+)-dependent nucleoside transport system could be inferred from the following observations: (a) Sodium depletion caused a strong inhibition of nucleoside transport reflected by an up to threefold and 15-fold increase in extracellular uridine and adenosine, respectively. The sodium-dependent transport of uridine was saturated when the influent uridine concentration was raised beyond 20 mumol/L. No such saturation was observed for much higher concentrations of adenosine used (10 to 75 mumol/L). (b) Na(+)-free perfusion resulted in a strong suppression of the release of uridine catabolites by the liver. Complete uridine breakdown was depressed to 7% of the amount of 14CO2 released in the presence of sodium and at influent uridine concentrations below 20 mumol/L. (c) Inhibition of uridine (10 mumol/L) transport and degradation was observed after coperfusion with adenosine, deoxyadenosine, guanosine and deoxyguanosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous adenosine inhibits hippocampal CA1 neurones: further evidence from extra- and intracellular recording.

Extracellular and intracellular recordings from CA1 pyramidal neurones of rats in vitro were used to study the effects of endogenous and exogenously applied adenosine. The adenosine receptor antagonist, caffeine, enhanced the intracellular recorded e.p.s.p.-i.p.s.p. sequence evoked by stimulation of the stratum radiatum which is antagonized by exogenous adenosine. The late, potassium dependent i.p.s.p. was not antagonized. The adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI), mimicked the effects of exogenously applied adenosine. The effects of NBTI and of exogenously applied adenosine were antagonized by caffeine in the same manner. Exposure to adenosine deaminase enhanced the evoked field e.p.s.p. During this enhancement caffeines effects were significantly reduced. In low calcium high magnesium medium which abolishes synaptic activity, adenosine deaminase increased, NBTI decreased cell firing. We conclude that endogenous adenosine, release by a calcium independent mechanism, can exert an inhibitory tone on CA1 neurones in vitro. This is consistent with a role for adenosine as a mediator of negative feedback between the metabolic state and electrophysiological activity of nervous tissue.

[3H]nitrobenzylthioinosine binding to the guinea pig CNS nucleoside transport system: a pharmacological characterization.

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific membrane sites in guinea pig brain was rapid, reversible, and saturable, and was dependent upon protein concentration, pH, and temperature. Mass law analysis of the binding data for cortical membranes indicated that NBMPR bound with high affinity to a single class of sites at which the equilibrium dissociation constant (KD) for NBMPR was 0.10-0.25 nM and which possessed a maximum binding capacity (Bmax) per mg of protein of 300 fmol of NBMPR. Kinetic analysis of the site-specific binding of NBMPR yielded an independent estimate of the KD of 0.16 nM. A relatively homogeneous subcellular distribution of the sites for NBMPR was found in cortical tissue. Recognized inhibitors of nucleoside transport were potent, competitive inhibitors of the binding of NBMPR in guinea pig CNS membranes whereas benzodiazepines and phenothiazines have low affinity for the sites. NBMPR sites in guinea pig cortical membranes have characteristics similar to those for NBMPR in human erythrocytes, the occupation of which is associated with inhibition of nucleoside transport. The comparable affinities for a range of agents for sites in human erythrocytes and guinea pig CNS membranes suggest that NBMPR also binds to transport inhibitory elements of the guinea pig CNS nucleoside transport system. It is proposed that the study of the binding of NBMPR provides an effective method by which to examine drug interactions with the membrane-located nucleoside transport system in CNS membranes.

Therapy of mouse leukemia L1210 with combinations of nebularine and nitrobenzylthioinosine 5'-monophosphate.

Earlier reports from this laboratory showed that: (a) in the presence of nitrobenzylthioinosine (NBMPR), a potent, tightly bound inhibitor of nucleoside transport, cells proliferating in culture were protected against a number of cytotoxic nucleosides; and (b) mice were protected against potentially lethal dosages of nebularine (and other toxic nucleosides) by coadministration of NBMPR. The present study, which used nitrobenzylthioinosine 5'-phosphate (NBMPR-P), a readily soluble "prodrug" form of NBMPR, extended the in vivo protection studies and showed that the half-life of the protection effect was about 4 hr. In chemotherapy experiments, mice bearing transplanted neoplasms were treated with high dosages of nebularine together with protecting doses of NBMPR-P. When mice bearing leukemia L1010 were treated with a potentially lethal regimen of nebularine administered together with NBMPR-P, a substantial kill of leukemic cells resulted (some mice were long-term survivors). The therapeutic effect was optimal at dosage levels of the protecting agent in excess of those required in nonleukemic mice for protection against the lethal nebularine dosages used, suggesting that the therapeutic effect was due to the joint presence in the leukemic cells of a metabolite of NBMPR-P and nebularine; NBMPR-P protection of the leukemic host against nebularine lethality was necessary for the therapeutic effect to be manifested.