Leishmania donovani: characteristics of adenosine and inosine transporters in promastigotes of two different strains

The nucleoside transport characteristics of two strains of Leishmania donovani promastigotes were studied. Strain S1, growing in fully defined medium, and strain S2 (MHOM/ET/67/HA3) both transported adenosine and inosine, but only strain S1 transported uridine and thymidine. Competition studies in the presence of 100 microM of unlabeled adenosine, inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, 3'-deoxyinosine as well as uridine, thymidine and cytidine, with either 1 microM [3H]adenosine or [3H]inosine as permeant, were carried out. The inhibition profile with [3H]inosine as permeant was essentially identical in S1 and S2 promastigotes, indicating that the same inosine transporter was present in both strains. However, with [3H] adenosine as permeant, significant differences were noted between the two strains. Thus, only adenosine, 2'-deoxyadenosine, tubercidin, uridine, and thymidine were strongly inhibitory in S1 promastigotes, while essentially all nucleosides tested were effective in S2 promastigotes. This indicates that adenosine transport in S2 promastigotes seems to involve a transporter differing from that described for S1 promastigotes.     

Chemical and genetic comparison of the glucose and nucleoside transporters

Glucose and nucleoside uptake into human red cells occurs through protein(s) which copurify in a complex, known as band 4.5 of relative mass (Mr) 66,000 to 50,000. The specific inhibitor of glucose transport, [3H]cytochalasin B, and the specific inhibitor of nucleoside transport, [3H]nitrobenzylthioribofuranosylpurine ([3H]NBMPR), incorporate covalently into component(s) of band 4.5 upon irradiation with ultraviolet light. Both photolabelled components are shown to be glycoproteins, since their migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is increased after treatment of photolabelled band 4.5 with endoglycosidase F. Peptide maps of the photolabelled components were compared. Red cell membranes were photolabelled with either [3H]cytochalasin B or [3H]NBMPR and subjected to SDS-PAGE. The region containing band 4.5 was cut and transferred to a second SDS-PAGE system and exposed to either papain or Staphylococcus aureus V8 protease. Papain (5 micrograms) completely cleaved band 4.5 and produced fragments of Mr 33,000, 26,000, 21,000, 15,000, and 12,500. Of these, the 21,000 fragment was the most conspicuous and it retained the label of [3H]cytochalasin B; the 33,000 fragment retained the label of [3H]NBMPR. The V8 protease (0.75 microgram) completely cleaved band 4.5 and produced fragments of Mr 35,000, 28,000, 22,000, 16,000, 13,500, and 9,000. The 28,000 fragment retained the label of [3H]cytochalasin B. The label of [3H]NBMPR was distributed along the gel in several regions comprising the 35,000, 28,000, and 16,000 fragments. Longer treatment with the V8 protease did not alter the position of the 28,000 [3H]cytochalasin B labelled peak, but completely abolished the [3H]NBMPR labelled peaks. Genetic segregation of the glucose and nucleoside transporters was determined in a lymphoma cell line. A mutant (14T- g) of S49 cells was selected which had lost the capacity to transport thymidine or to bind NBMPR. Uptake of either 2-deoxyglucose or 3-O-methylglucose, inhibitable by cytochalasin B, was not impaired in this mutant. It is concluded that the nucleoside and glucose transporters are glycoprotein components of band 4.5, which are differentiated by peptide map analysis. Further, a lymphoblast mutant was isolated which had lost the nucleoside transport function but retained the glucose transport function.     

Involvement of adenosine kinase in the phosphorylation of formycin B in CHO cells

In Chinese hamster ovary cells, [3H]formycin B is metabolized into formycin B-5'-monophosphate, formycin A-5'-monophosphate and higher phosphorylated derivatives of formycin A which are incorporated into RNA. Mutants of CHO cells independently selected for resistance to various adenosine analogs viz. toyocamycin, tubercidin, 6-methylmercaptopurine riboside, which contain no detectable activity of adenosine kinase (AK) in cell extracts, all exhibited between 2- to 3-fold increased resistance to formycin B. Formycin B-resistant mutants of CHO cells are also affected in AK, as indicated by the absence of AK activity in cell extracts. Both types of AK- mutants showed reduced uptake and phosphorylation of [3H]formycin B in comparison to the parental (AK+) cells. In addition, toxicity of formycin B towards CHO cells was reduced in presence of adenosine in a concentration dependent manner. These observations strongly indicate that in CHO cells, formycin B is phosphorylated via AK and that like other nucleoside analogs its phosphorylation may be essential for the drugs cellular toxicity.     

Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine

The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2'-3H]-adenosine, there was a 8.9% release of the C-2' tritium from [2'-3H]adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.     

Quantitative Analysis of Similarities and Differences in Neurotoxicities Caused by Adenosine and 2'-Deoxyadenosine in Sympathetic Neurons

Abstract: These experiments characterize the nucleoside transport and quantify the neurotoxicity of adenosine and 2′-deoxyadenosine (dAdo) in chick sympathetic neurons. We show that [³H]adenosine transport was sensitive to low temperature, specific inhibitors of nucleoside transport, and an excess concentration of adenosine. However, many of these treatments had a marginal effect on [³H]dAdo transport. Total retention of [³H]dAdo over short and long periods was ～10 times less than that of [³H]adenosine. These data suggest that adenosine and dAdo enter sympathetic neurons by different routes. Uptake of [³H]norepinephrine ([³H]NE) decreased in neurons damaged by nucleosides and increased to control levels when neurons were protected by various agents against adenosine or dAdo toxicity. These results indicate that [³H]NE uptake serves as a quantitative index of toxicity by the nucleosides. Using this approach we demonstrate that phosphorylation of both nucleosides is essential for their lethal action. For example, iodotubercidin prevented nucleoside-induced neuronal death, but the effect was much more pronounced in the case of dAdo toxicity (IC₅₀ of 0.83 ± 0.4 vs. 30 ± 1.6 nM). Another kinase inhibitor, 5′-amino 5′-deoxyadenosine, was effective in protecting neurons against dAdo but had no effect against adenosine toxicity. These results suggest that specific kinases are associated with the phosphorylation of adenosine and dAdo in sympathetic neurons to produce toxic metabolic products. Finally, neurons were susceptible to dAdo toxicity from the time of plating to 4 weeks in culture but were resistant to adenosine toxicity 8 h after plating. In conclusion, our results highlight major differences in the mechanism of neurotoxicity by adenosine and dAdo and provide insights for identification of biochemical pathways leading to neuronal death.     

Involvement of Bidirectional Adenosine Transporters in the Release of l-[3H]Adenosine from Rat Brain Synaptosomal Preparations

Abstract: Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with l -[³H]adenosine, our objectives here were to determine (1) if l -[³H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological d -adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. l -[³H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. l -[³H]Adenosine release was inhibited dose-dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 µM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with l -[³H]adenosine alone or l -[³H]adenosine plus unlabeled l -adenosine, d -adenosine, or uridine, l -[³H]-adenosine release was inhibited 42% by l -adenosine, 69% by uridine, and 81% by d -adenosine. The inhibition of l -[³H]adenosine release from the synaptosomal preparations by substrates for or inhibitors of nucleoside transporters suggests that a portion of the release was mediated by nucleoside transporters. This experimental system may prove useful for evaluating the effects of pharmacological agents on bidirectional transport of adenosine.     

Sodium-Dependent Uptake of Nucleosides by Dissociated Brain Cells from the Rat

Sodium-dependent 3H-labeled nucleoside transport was studied using a mixed population of dissociated brain cells from adult rats. The accumulation of [3H]adenosine during brief (15-s) incubation periods was significantly greater in the presence of 110 mM Na+ than in its absence. This occurred at substrate concentrations that ranged from 0.25 to 100 microM. Similar findings were observed for the rapid accumulation of [3H]uridine. Kinetically, the rapid accumulation of [3H]adenosine in both the absence and the presence of Na+ was best described by a two-component system. In the presence of Na+, the KT and Vmax values for the high-affinity affinity component were 0.9 microM and 8.9 pmol/mg of protein/15 s, and those for the low-affinity component were 313 microM and 3,428 pmol/mg of protein/15 s, respectively. In the absence of Na+, the KT value for the high-affinity component was significantly higher (1.8 microM). [3H]Uridine accumulation was best described kinetically by a one-component system that in the presence of Na+ had KT and Vmax values of 1.0 mM and 2.6 nmol/mg of protein/15 s, respectively. As was found for [3H]adenosine, in the absence of Na+, the KT value was significantly higher (1.8 mM). The sodium-dependent transport of [3H]adenosine was inhibitable by ouabain and 2,4-dinitrophenol. Of the three nucleoside transport inhibitors tested, only nitrobenzylthioninosine demonstrated high affinity and selectivity in blocking the sodium component. Thus, high-affinity sodium-dependent nucleoside transport systems, in addition to facilitated diffusion systems, exist on brain cells from adult rats.     

Identification of the nucleoside transporter in cultured mouse lymphoma cells. Photoaffinity labeling of plasma membrane-enriched fractions from nucleoside transport-competent (S49) and nucleoside transport-deficient (AE1) cells with [3H]nitrobenzylthioinosine

Plasma membrane-enriched fractions from disrupted S49 lymphoma cells contained high affinity sites for [3H]nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport. These sites were absent from similar preparations from AE1 cells, a nucleoside-transport deficient clone derived from the S49 cell line. Reversible binding of [3H]nitrobenzylthioinosine to the S49 membrane preparations was inhibited by adenosine, nitrobenzylthioguanosine, and dipyridamole. Exposure of S49 membrane preparations to UV light in the presence of [3H]nitrobenzylthioinosine resulted in the covalent radiolabeling of a membrane protein(s) which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of 45,000 to 66,000. Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine and markedly reduced in the presence of adenosine and dipyridamole. AE1 membrane proteins were not covalently labeled under these conditions.     

Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V(max) of 3.4 +/- 1 pmol.microl(-1).s(-1), with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs (K(m) = 96 +/- 37 microM; V(max) = 1.6 +/- 0.3 pmol.microl(-1).s(-1)) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine (K(i) = 19 +/- 7 microM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK(15) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na(+) or H(+). PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.     

Isolation and characterization of an L1210 cell line retaining the sodium-dependent carrier cif as its sole nucleoside transport activity

Nucleoside permeation across mammalian cell membranes is complex with at least four distinct transporters known. Two of these (es and ei) are equilibrative (facilitated diffusion) carriers that have been studied is considerable detail. The other two (cif and cit) are concentrative, Na(+)-dependent carriers. A major obstacle to the characterization of the latter two mechanisms has been the lack of suitable model systems expressing only a single nucleoside transport activity. The present study describes the isolation of a cell line that has cif as its sole nucleoside transporter. L1210/MC5-1 cells, which have es and cif transport activity, were mutagenized and plated in soft agar containing two cytotoxic nucleosides (tubercidin (7-deazaadenosine) and cytosine arabinoside) that are substrates for es but not cif. A clonal line (L1210/MA-27.1) was isolated which retained the capacity for Na(+)-dependent [3H]formycin B transport but was unable to transport [3H]thymidine, a substrate for es but not cif. Failure of the mutant to transport thymidine was also demonstrated by the inability of thymidine (with adenine as a purine source) to rescue these cells from methotrexate toxicity. Furthermore, the mutant lacked nitrobenzylthioinosine (NBMPR) binding activity (an integral part of the es transporter) as demonstrated by reversible NBMPR binding and photoaffinity labeling with [3H]NBMPR. Loss of es transport activity was also demonstrated by the failure of NBMPR to affect the toxicity of 2-chlorodeoxyadenosine (IC50 approximately 30 nM) in L1210/MA27.1 cells. In contrast, NBMPR decreased the IC50 for 2-chlorodeoxyadenosine from 100 to 30 nM in the parental L1210/MC5-1 cell line. These results are consistent with the mechanism of NBMPR potentiation of 2-chlorodeoxyadenosine toxicity in L1210 cells being a blockade of efflux via es while the nucleoside is pumped into the cells by the concentrative cif carrier.     

Effect of 5''-(N-Ethylcarboxamido)adenosine on Adenosine Transport in Cultured Chromaffin Cells

Extracellular adenosine is transported into chromaffin cells by a high-affinity transport system. The action of adenosine receptor ligands was studied in this cellular model. 5'-(N-Ethylcarboxamido)adenosine (NECA), an agonist of A2 receptors, activated adenosine transport. Km values for adenosine were 4.6 +/- 1.0 (n = 5) and 10.2 +/- 3.0 microM (n = 5) for controls and 100 nM NECA, respectively. The Vmax values were 66.7 +/- 23.5 and 170.2 +/- 30 pmol/10(6) cells/min for controls and 100 nM NECA, respectively. The A1 agonist N6-cyclohexyladenosine, the A1 antagonist 8-cyclopentyl-1, 3-dipropylxanthine, and the A1-A2 antagonist 1,3-dipropyl-8-(4-[(2-aminoethyl)amino]-carbonylmethyloxyphenyl)- xanthine did not significantly modify the adenosine transport in this system. Binding studies done with [3H]dipyridamole, a nucleoside transporter ligand, did not show changes in either the number or affinity of transporter sites after NECA treatment. This ligand can enter cells and quantifies the total number of transporters. The binding studies with [3H]-nitrobenzylthioinosine, which quantifies the plasma membrane transporters, showed a Bmax of 19,200 +/- 800 and 23,200 +/- 700 transporters/cell for controls and 100 nM NECA, respectively. No changes in the KD were obtained. The effects of NECA were not mediated through adenylate cyclase activation, because its action was not imitated by forskolin.     

Human red-blood-cell Ca2+-antagonist binding sites. Evidence for an unusual receptor coupled to the nucleoside transporter

The human red blood cell ghost Ca2+-antagonist binding sites were characterized with (+/-)-[3H]nimodipine. The labelled 1,4-dihydropyridine bound in a non-cooperative, reversible manner with a Kd of 52 nM at 25 degrees C to 9.65 pmol sites/mg ghost protein. The stereochemistry of the binding domain was evaluated with the optically pure enantiomers of chiral 1,4-dihydropyridines. In contrast to the 1,4-dihydropyridine-selective receptors on Ca2+ channels in electrically excitable tissues, the (+) enantiomer of nimodipine and the (-) enantiomer of the benzoxadiazol 1,4-dihydropyridine (PN 200-110) were bound with higher affinity than the respective optical antipodes. The human red blood cell ghost [3H]nimodipine-labelled sites also interacted with the inorganic Ca2+-antagonist La3+ (increase in the number of binding sites), and were allosterically regulated by the optical enantiomers of the phenylalkylamine-type Ca2+-antagonists (e.g. verapamil, desmethoxyverapamil, methoxyverapamil). The benzothiazepines d- or l-cis-diltiazem were without effect. Nucleosides (adenosine approximately equal to inosine greater than cytidine) were inhibitory at the nimodipine-labelled site, as were the nucleoside uptake inhibitors dipyridamole, hexobendine, dilazep, nitrobenzylthioinosine and nitrobenzylthioguanosine. The binding sites have essential sulfhydryl groups, show trypsin sensitivity, but are relatively heat stable. When nitrobenzylthioinosine was employed as a covalent probe to inactivate the red blood cell ghost nucleoside carrier, [3H]nimodipine binding was irreversibly lost. (+)-Nimodipine greater than (-)-nimodipine inhibited [14C]adenosine transport into human red blood cells. A good correlation between IC50 values for inhibition of [3H]nimodipine binding and IC50 values for inhibition of [14C]adenosine uptake was found for 18 compounds. Sheep red blood cells (which lack the nucleoside transporter) had no detectable [3H]nimodipine binding sites. It is concluded that the Ca2+-antagonist receptor sites of the human erythrocyte are coupled to the nucleoside transporter.     

Nucleoside transport in heart: species differences in nitrobenzylthioinosine binding, adenosine accumulation, and drug-induced potentiation of adenosine action

The site-specific binding of the potent and selective nucleoside transport inhibitor, [3H]nitrobenzylthioinosine (NBMPR), to the nucleoside transport system of cardiac membranes of several species was investigated. The affinity of [3H]NBMPR for these sites ranged from 0.03 nM in rat to 0.78 nM in dog. The maximal binding capacity of cardiac membranes for [3H]NBMPR was also species dependent and was greatest in bovine and guinea pig heart (2551 and 1700 fmol/mg protein, respectively) and least in rat (195 fmol/mg protein). The affinities of recognized nucleoside transport inhibitors and benzodiazepines for these transport inhibitory sites in guinea pig and rat heart were estimated by studying the inhibition of the site-specific binding of [3H]NBMPR in competition experiments. These values were compared with their inhibitory effects on the transporter-dependent accumulation of [3H]adenosine in guinea pig and rat cardiac muscle segments and with their ability to potentiate the negative inotropic action of adenosine in electrically driven guinea pig and rat left atria. In guinea pig heart, the recognized nucleoside transport inhibitors and benzodiazepines had an order of affinity (dilazep greater than hydroxynitrobenzylthioguanosine greater than dipyridamole greater than hexobendine much greater than lidoflazine much greater than flunitrazepam greater than diazepam greater than lorazepam greater than flurazepam) for the NBMPR site which was similar to those for the inhibition of [3H]adenosine accumulation and for potentiation of adenosine action. In contrast, in rat heart, where the maximal binding capacity of [3H]NBMPR was lower (eightfold), the nucleoside transporter dependent accumulation of [3H]adenosine was also lower (sixfold) and the negative inotropic action of adenosine was not significantly potentiated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine and tubercidin binding and transport in Chinese hamster ovary and Novikoff rat hepatoma cells

The uptake of adenosine and tubercidin by control and ATP-deleted wild-type and adenosine kinase-deficient cells was measured by rapid kinetic techniques. Adenosine deamination was inhibited by pretreatment with 2-deoxy-coformycin. Control wild-type cells phosphorylated adenosine so rapidly that the kinetics of transport per se could not be assessed unambiguously. ATP depletion and adenosine kinase deficiency did not abolish the conversion of adenosine to nucleotides, but reduced it to such an extent that initial velocities of uptake could be safely construed as transport velocities in both zerotrans and equilibrium exchange modes. The same was true for tubercidin, which was not phosphorylated in adenosine kinase-deficient cells. It accumulated intracellularly, however, to concentrations 50 to 120% higher than those in the extracellular space, apparently due to binding to some intracellular component(s). Binding was not saturated up to a concentration of 200 μM, but seemed to be slow relative to transport. Fits of appropriate integrated rate equations based on the simple carrier model to uptake time courses obtained under these conditions yielded Michaelis-Menten constants for adenosine and tubercidin transport of 100 to 200 μM and maximum velocities of 10 to 30 pmol/μl cell H₂O ? sec, whereas the rate of intracellular phosphorylation was maximal at concentrations between 2 and 8 μM. The first-order rate constant (V_max/K_m) for adenosine phosphorylation, however, seemed to be appreciably higher than that for its transport. This indicates that at physiological concentrations, which fall in the first-order range for both processes, adenosine trapping is very efficient. Adenosine, tubercidin, tricyclic nucleoside, 2′-deoxyadenosine, and 3′-deoxyadenosine all inhibited uridine and thymidine transport to about the same extent, whereas pyrazofurin was signficantly less effective.     

Adenosine transport by the lung

Adenosine, a nucleoside and potent vasodilator, has been found to be taken up by the lung and converted by deamination into inosine and hypoxanthine. In a single circulation through an isolated rat lung, 69.3 +/- 3.3% of infused [14C]adenosine (10 microM) was removed from the circulation. Uptake of [14C]adenosine remained unchanged when deamination of adenosine was inhibited by 8-azaguanine or coformycin. In a single passage of adenosine through the pulmonary artery, very little of the deaminated products appeared in the pulmonary circulation, but when adenosine was recirculated through the pulmonary circulation inosine and hypoxanthine appeared in the venous effluent. These adenosine metabolites were also taken up by the lung. A major portion of the circulating adenosine was transported into the lung, where it was used to synthesize adenine nucleotides. Inhibition of adenosine kinase by iodotubercidin resulted in reduced formation of ATP and ADP. Uptake of adenosine by the lung was saturable on a concentration gradient and was a passive process because it was not affected by the absence of glucose or the presence of ouabain. Km and Vmax for adenosine transport were 0.227 mM and 4.6 mumol.min-1.g lung-1, respectively. Adenosine transport was inhibited by adenosine analogues, and the inhibitions were found to be competitive in nature. These results suggest that a specific and rate-limiting transport system exists in the lung for adenosine.     

Absence of binding sites for the transport inhibitor nitrobenzylthioinosine on nucleoside transport-deficient mouse lymphoma cells

Cells of an adenosine-resistant clone (AE₁) of S49 mouse lymphoma cells were compared with cells of the parental line with respect to (a) characteristics of nucleoside transport, (b) high affinity binding of the inhibitor of nucleoside transport, nitrobenzylthionisine (NBMPR), and (c) the antiproliferative effects of the nucleoside antibiotics, tubercidin, arabinosyladenine and showdomycin. Rates of inward transport of uridine, thymidine, adenosine, 2′-deoxyadenosine, tubercidin, showdomycin, and arabinosyladenine in AE₁ cells were less than 1% of those in cells of the parental S49 line. The inhibitor of nucleoside transport, NBMPR, reduced rates of inward nucleoside transport in S49 cells to levels comparable to those seen in the transport-defective mutant. S49 cells possessed high affinity sites that bound NBMPR (6.6 · 10⁴ sites/cell, K_d  0.2 nM), whereas site-specific binding of NBMPR to AE₁ cells was not demonstrable, indicating that loss of nucleoside transport activity in AE₁ cells was accompanied by loss of the high affinity NBMPR binding sites. Relative to S49 cells, AE₁ cells were resistant to the antiproliferative effects of tubercidin and showdomycin, but differences between the two cell lines in sensitivity toward arabinosyladenine were minor, suggesting that nucleoside transport activity was required for cytotoxicity of tubercidin and showdomycin, but not for that of arabinosyladenine.     

Functional and molecular characterization of adenosine transport at the rat inner blood-retinal barrier

The purpose of the present study was to characterize the adenosine transport system(s) at the inner blood-retinal barrier (inner BRB). A conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2), used as an in vitro model of the inner BRB, expresses equilibrative nucleoside transporter 1 (ENT1), ENT2, concentrative nucleoside transporter 2 (CNT2), and CNT3 mRNAs. TR-iBRB2 cells exhibited an Na⁺-independent and concentration-dependent [³H]adenosine uptake with a Michaelis-Menten constant of 28.5 μM and a maximum uptake rate of 814 pmol/(min mg protein). [³H]Adenosine uptake by TR-iBRB2 cells was strongly inhibited by 2 mM adenosine, inosine, uridine, and thymidine. On the other hand, this process was not inhibited by 100 nM nitrobenzylmercaptopurine riboside and dipyridamole. These uptake studies suggest that ENT2 is involved in [³H]adenosine uptake by TR-iBRB2 cells. Quantitative real-time PCR revealed that the expression of ENT2 mRNA is 5.5-fold greater than that of ENT1 mRNA. An in vivo study suggested that [³H]adenosine is transported from the blood to the retina and significantly inhibited by adenosine and thymidine. The results of this study show that ENT2 most likely mediates adenosine transport at the inner BRB and is expected to play an important role in regulating the adenosine concentration in the retina.     

Cytoplasmic location of undermethylated messenger RNA in Novikoff cells.

Novikoff cells in culture were labeled with L-[methyl-3H]methionine and [U-14C]uridine in the presence of (a) TubHcy2, (b) AdoHcy, (c) homocysteine, (d) tubercidin, or (e) without any additions. Only in cultures labeled in the presence of TubHcy were undermethylated cap structures observed to represent a significant portion of [3H]methyl radioactivity. Novikoff cells in culture were then simultaneously labeled with L-[methyl-3H]methionine and [32P]orthophosphate in the presence or absence of TubHcy. Total cytoplasmic, polysomal and monosomal poly(A)-containing RNAs were analyzed. Both monosomal and polysomal mRNA fractions from TubHcy-treated cells contain partially methylated cap structures, suggesting that 2'-O-methylation of the nucleoside adjacent to the pyrophosphate linkage in caps is not required for transport, ribosomal binding or translation. Comparison of nuclear and cytoplasmic cap structures from normal and inhibited cultures indicate that an altered mRNA population is generated in the presence of TubHcy.     

Regional distribution of adenosine uptake in guinea-pig brain slices and the effect of some inhibitors: Evidence for nitrobenzylthioinosine-sensitive and insensitive sites?

The accumulation of [2-³H]adenosine was measured in slices prepared from 7 regions of the guinea-pig central nervous system. There was a similar level of uptake in forebrain regions (cerebral cortex, striatum, hippocampus and midbrain), a lower level in the cerebellum, with lowest uptake in the pons-medulla and spinal cord. Uptake in all regions was strongly inhibited by the nucleoside transport inhibitor dipyridamole and by 5-iodotubercidin, an adenosine kinase inhibitor. The activity of adenosine kinase was similar in crude supernatants prepared from 8 regions of the guinea-pig and rat brain, with the exception of the spinal cord (lower activity than other regions in the guinea-pig CNS) and olfactory bulb (higher activity than other regions in the rat CNS). 5-Nitrobenzylthioinosine (NBMPR) and related thiopurines produced about 50% inhibition of adenosine uptake into guinea-pig cerebral cortex slices at 200 nM but increasing the concentration did not produce significant further inhibition. [³H]NBMPR has been proposed as a useful tight-binding ligand for nucleoside transport sites in various tissues but it is suggested that the distribution of such binding sites in different regions of the CNS may not directly reflect the adenosine uptake capacity of these regions1. Data suggest that there may be NBMPR-sensitive and -insensitive sites. Results confirm those of previous studies which suggest that intracellular adenosine kinase plays an important part in the uptake of adenosine in guinea-pig brain. The relatively homogeneous distribution of adenosine uptake activity in the brain contrasts with the heterogeneous distribution of A1-adenosine receptors in the CNS.     

Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.