Mild hypothermia protects synaptic transmission from experimental ischemia through reduction in the function of nucleoside transporters in the mouse hippocampus.

Ischemia, a severe metabolic stress, increases adenosine levels and causes the suppression of synaptic transmission through adenosine A1 receptors. Although temperature also regulates extracellular adenosine levels, the effect of temperature on ischemia-induced activation of adenosine receptors is not yet fully understood. Here we examined the role of adenosine A1 receptors in mild hypothermia-mediated neuroprotection during the acute phase of ischemia. Severe ischemia-induced neurosynaptic impairment was reproduced by oxygen-glucose deprivation at normothermia (36 °C) and assessed with extracellular recordings or whole-cell patch clamp recordings in acute hippocampal slices in mice. Mild hypothermia (32 °C) induced the protection of synaptic transmission by activating adenosine A1 receptors. Stricter hypothermia (28 °C) caused additional neuroprotective effects by extending the onset time to anoxic depolarization; however, this effect was not associated with adenosine A1 receptors. The response of exogenous adenosine-induced inhibition of hippocampal synaptic transmission was increased by lowering the temperature to 32 °C or 28 °C. Hypothermia also reduced the function of dipryidamole-sensitive nucleoside transporters. These findings suggest that an increased response of adenosine A1 receptors, caused by a reduction in the function of nucleoside transporters, is one mechanism by which therapeutic hypothermia (usually used within the mild range) mediates neurosynaptic protection in the acute phase of stroke.

The Adenosinergic System as a Therapeutic Target in the Vasculature: New Ligands and Challenges.

Adenosine is an adenine base purine with actions as a modulator of neurotransmission, smooth muscle contraction, and immune response in several systems of the human body, including the cardiovascular system. In the vasculature, four P1-receptors or adenosine receptors-A1, A2A, A2B and A3-have been identified. Adenosine receptors are membrane G-protein receptors that trigger their actions through several signaling pathways and present differential affinity requirements. Adenosine is an endogenous ligand whose extracellular levels can reach concentrations high enough to activate the adenosine receptors. This nucleoside is a product of enzymatic breakdown of extra and intracellular adenine nucleotides and also of S-adenosylhomocysteine. Adenosine availability is also dependent on the activity of nucleoside transporters (NTs). The interplay between NTs and adenosine receptors' activities are debated and a particular attention is given to the paramount importance of the disruption of this interplay in vascular pathophysiology, namely in hypertension., The integration of important functional aspects of individual adenosine receptor pharmacology (such as in vasoconstriction/vasodilation) and morphological features (within the three vascular layers) in vessels will be discussed, hopefully clarifying the importance of adenosine receptors/NTs for modulating peripheral mesenteric vascular resistance. In recent years, an increase interest in purine physiology/pharmacology has led to the development of new ligands for adenosine receptors. Some of them have been patented as having promising therapeutic activities and some have been chosen to undergo on clinical trials. Increased levels of endogenous adenosine near a specific subtype can lead to its activation, constituting an indirect receptor targeting approach either by inhibition of NT or, alternatively, by increasing the activity of enzymes responsible for ATP breakdown. These findings highlight the putative role of adenosinergic players as attractive therapeutic targets for cardiovascular pathologies, namely hypertension, heart failure or stroke. Nevertheless, several aspects are still to be explored, creating new challenges to be addressed in future studies, particularly the development of strategies able to circumvent the predicted side effects of these therapies.

Equilibrative nucleoside transporters-A review.

Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that mediate the transport of nucleosides, nucleobases, and therapeutic analogs. The best-characterized ENTs are the human transporters hENT1 and hENT2. However, non-mammalian eukaryotic ENTs have also been studied (e.g., yeast, parasitic protozoa). ENTs are major pharmaceutical targets responsible for modulating the efficacy of more than 30 approved drugs. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. This review highlights findings on the characterization of ENTs by surveying studies on genetics, permeant and inhibitor interactions, mutagenesis, and structural models of ENT function.

Guanosine protects glial cells against 6-hydroxydopamine toxicity.

Increasing body of evidence indicates that neuron-neuroglia interaction may play a key role in determining the progression of neurodegenerative diseases including Parkinson's disease (PD), a chronic pathological condition characterized by selective loss of dopaminergic (DA) neurons in the substantia nigra. We have previously reported that guanosine (GUO) antagonizes MPP(+)-induced cytotoxicity in neuroblastoma cells and exerts neuroprotective effects against 6-hydroxydopamine (6-OHDA) and beta-amyloid-induced apoptosis of SH-SY5Y cells. In the present study we demonstrate that GUO protected C6 glioma cells, taken as a model system for astrocytes, from 6-OHDA-induced neurotoxicity. We show that GUO, either alone or in combination with 6-OHDA activated the cell survival pathways ERK and PI3K/Akt. The involvement of these signaling systems in the mechanism of the nucleoside action was strengthened by a reduction of the protective effect when glial cells were pretreated with U0126 or LY294002, the specific inhibitors of MEK1/2 and PI3K, respectively. Since the protective effect on glial cell death of GUO was not affected by pretreatment with a cocktail of nucleoside transporter blockers, GUO transport and its intracellular accumulation were not at play in our in vitro model of PD. This fits well with our data which pointed to the presence of specific binding sites for GUO on rat brain membranes. On the whole, the results described in the present study, along with our recent evidence showing that GUO when administered to rats via intraperitoneal injection is able to reach the brain and with previous data indicating that it stimulates the release of neurotrophic factors, suggest that GUO, a natural compound, by acting at the glial level could be a promising agent to be tested against neurodegeneration.

Modulation of adenosine signaling prevents scopolamine-induced cognitive impairment in zebrafish.

Adenosine, a purine ribonucleoside, exhibits neuromodulatory and neuroprotective effects in the brain and is involved in memory formation and cognitive function. Adenosine signaling is mediated by adenosine receptors (A1, A2A, A2B, and A3); in turn, nucleotide and nucleoside-metabolizing enzymes and adenosine transporters regulate its levels. Scopolamine, a muscarinic cholinergic receptor antagonist, has profound amnesic effects in a variety of learning paradigms and has been used to induce cognitive deficits in animal models. This study investigated the effects of acute exposure to caffeine (a non-selective antagonist of adenosine receptors A1 and A2A), ZM 241385 (adenosine receptor A2A antagonist), DPCPX (adenosine receptor A1 antagonist), dipyridamole (inhibitor of nucleoside transporters) and EHNA (inhibitor of adenosine deaminase) in a model of pharmacological cognitive impairment induced by scopolamine in adult zebrafish. Caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA were acutely administered independently via i.p. in zebrafish, followed by exposure to scopolamine dissolved in tank water (200µM). These compounds prevented the scopolamine-induced amnesia without impacting locomotor activity or social interaction. Together, these data support the hypothesis that adenosine signaling may modulate memory processing, suggesting that these compounds present a potential preventive strategy against cognitive impairment.

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells.

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2 × 7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2 × 7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2 × 7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling-p53 increase, AMPK activation, and PARP cleavage-as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.

Steroid hormones are novel nucleoside transport inhibitors by competition with nucleosides for their transporters.

Nucleoside transport is important for nucleic acid synthesis in cells that cannot synthesize nucleosides de novo, and for entry of many cytotoxic nucleoside analog drugs used in chemotherapy. This study demonstrates that various steroid hormones induce inhibition of nucleoside transport in mammalian cells. We analyzed the inhibitory effects of estradiol (E2) on nucleoside transport using SH-SY5Y human neuroblastoma cells. We observed inhibitory effects after acute treatment with E2, which lasted in the presence of E2. However, when E2 was removed, the effect immediately disappeared, suggesting that E2 effects are not mediated through the canonical regulatory pathway of steroid hormones, such as transcriptional regulation. We also discovered that E2 could competitively inhibit thymidine uptake and binding of the labeled nucleoside transporter inhibitor, S-[4-nitrobenzyl]-6-thioinosine (NBTI), indicating that E2 binds to endogenous nucleoside transporters, leading to inhibition of nucleoside transport. We then tested the effects of various steroids on nucleoside uptake in NBTI-sensitive cells, SH-SY5Y and NBTI-insensitive cells H9c2 rat cardiomyoblasts. We found E2 and progesterone clearly inhibited both NBTI-sensitive and insensitive uptake at micromolar concentrations. Taken together, we concluded that steroid hormones function as novel nucleoside transport inhibitors by competition with nucleosides for their transporters.

Erlotinib, gefitinib, and vandetanib inhibit human nucleoside transporters and protect cancer cells from gemcitabine cytotoxicity.

PURPOSE: Combinations of tyrosine kinase inhibitors (TKI) with gemcitabine have been attempted with little added benefit to patients. We hypothesized that TKIs designed to bind to ATP-binding pockets of growth factor receptors also bind to transporter proteins that recognize nucleosides. EXPERIMENTAL DESIGN: TKI inhibition of uridine transport was studied with recombinant human (h) equilibrative (E) and concentrative (C) nucleoside transporters (hENT, hCNT) produced individually in yeast. TKIs effects on uridine transport, gemcitabine accumulation, regulation of hENT1 activity, and cell viability in the presence or absence of gemcitabine were evaluated in human pancreatic and lung cancer cell lines. RESULTS: Erlotinib, gefitinib and vandetanib inhibited [(3)H]uridine transport in yeast and [(3)H]uridine and [(3)H]gemcitabine uptake in the four cell lines. Treatment of cell lines with erlotinib, gefitinib, or vandetanib for 24 hours reduced hENT1 activity which was reversed by subsequent incubation in drug-free media for 24 hours. Greater cytotoxicity was observed when gemcitabine was administered before erlotinib, gefitinib, or vandetanib than when administered together and synergy, evaluated using the CalcuSyn Software, was observed in three cell lines resulting in combination indices under 0.6 at 50% reduction of cell growth. CONCLUSIONS: Vandetanib inhibited hENT1, hENT2, hCNT1, hCNT2, and hCNT3, whereas erlotinib inhibited hENT1 and hCNT3 and gefitinib inhibited hENT1 and hCNT1. The potential for reduced accumulation of nucleoside chemotherapy drugs in tumor tissues due to inhibition of hENTs and/or hCNTs by TKIs indicates that pharmacokinetic properties of these agents must be considered when scheduling TKIs and nucleoside chemotherapy in combination.

Functional identification of the hypoxanthine/guanine transporters YjcD and YgfQ and the adenine transporters PurP and YicO of Escherichia coli K-12.

The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate selectivity. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The two pairs of paralogs differ clearly in their substrate and ligand preferences. Of 25 putative inhibitors tested, PurP and YicO recognize with low micromolar affinity N(6)-benzoyladenine, 2,6-diaminopurine, and purine, whereas YjcD and YgfQ recognize 1-methylguanine, 8-azaguanine, 6-thioguanine, and 6-mercaptopurine and do not recognize any of the PurP ligands. Furthermore, the permeases PurP and YjcD were subjected to site-directed mutagenesis at highly conserved sites of transmembrane segments 1, 3, 8, 9, and 10, which have been studied also in COG2233 homologs. Residues irreplaceable for uptake activity or crucial for substrate selectivity were found at positions occupied by similar role amino acids in the Escherichia coli xanthine- and uric acid-transporting homologs (XanQ and UacT, respectively) and predicted to be at or around the binding site. Our results support the contention that the distantly related transporters of COG2233 and COG2252 use topologically similar side chain determinants to dictate their function and the distinct purine selectivity profiles.

Dipyridamole analogs as pharmacological inhibitors of equilibrative nucleoside transporters. Identification of novel potent and selective inhibitors of the adenosine transporter function of human equilibrative nucleoside transporter 4 (hENT4).

To identify needed human equilibrative nucleoside transporter 4 (hENT4) inhibitors, we cloned and stably expressed the recombinant protein in PK15NTD (nucleoside transporter deficient) cells, and, investigated its interaction with a series of dipyridamole analogs synthesized in our laboratory. Compounds were tested in this newly established hENT4 expressing system as well in previous stably expressed hENT1 and hENT2 expressing systems. Of the dipyridamole analogs evaluated, about one fourth of the compounds inhibited hENT4 with higher potencies than dipyridamole. The most potent of them, Compound 30 displayed an IC50 of 74.4 nM, making it about 38 times more potent than dipyridamole (IC50=2.8 µM), and selectivities of about 80-fold and 20-fold relative to ENT1 and ENT2, respectively. Structure-activity relationship showed nitrogen-containing monocyclic rings and noncyclic substituents at the 4- and 8-positions of the pyrimido[5,4-d]pyrimidine were important for the inhibitory activity against hENT4. The most potent and selective hENT4 inhibitors tended to have a 2,6-di(N-monohydroxyethyl) substitution on the pyrimidopyrimidine ring system. The inhibitors of hENT4 identified in this study are the most selective and potent inhibitors of hENT4 adenosine transporter function to date, and should serve as useful pharmacological/biochemical tools and/or potential leads for ENT4-based therapeutics. Also, the new hENT4-expressing PK15 cell line established will serve as a useful screening tool for the discovery and design of hENT4 ligands.

Comparison of uptake mechanisms for anthracyclines in human leukemic cells.

AIMS: The mechanisms behind cellular anthracycline uptake are not completely understood. Knowledge about uptake mechanisms could be used to increase the selectivity of the drugs. We compared the uptake patterns of, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and pirarubicin (PIRA) by cultured leukemic cells and investigated possible involvement of specific carriers. METHODS: HL-60 cells were incubated with anthracyclines for 1 hour in the absence or presence of transport inhibitors, suramin, or nucleosides and cellular drug uptake was determined. Cell survival was also determined. MCF-7 breast cancer cells were used as a negative control for concentrative nucleoside transporters (CNTs). Anthracycline concentration was determined with HPLC and fluorometric detection and apoptosis was determined with propidium iodide and flow cytometry. RESULTS: DNR, IDA, and PIRA had higher uptake than DOX and EPI with a prominent increase in uptake at concentrations > 1 µM. Uptake of all anthracyclines was greatly reduced at 0°C. Suramin, a purinergic-2-receptor inhibitor, strongly inhibited the uptake of all anthracyclines except PIRA and increased cell survival. Dipyridamole, an equilibrative NT (ENT) inhibitor, significantly inhibited the uptake of DNR only. The addition of nucleosides significantly inhibited the uptake of DNR, IDA, and PIRA but not in MCF-7 cells lacking functional CNTs. CONCLUSION: Our results suggest different uptake mechanisms for the anthracyclines studied. We found evidence for carrier mediated uptake mechanisms, supporting involvement of NTs in transmembrane transport of DNR, IDA, and PIRA. The results also showed a strong inhibition of suramin on anthracycline uptake by so far unknown mechanisms.

Discovering thiamine transporters as targets of chloroquine using a novel functional genomics strategy.

Chloroquine (CQ) and other quinoline-containing antimalarials are important drugs with many therapeutic benefits as well as adverse effects. However, the molecular targets underlying most such effects are largely unknown. By taking a novel functional genomics strategy, which employs a unique combination of genome-wide drug-gene synthetic lethality (DGSL), gene-gene synthetic lethality (GGSL), and dosage suppression (DS) screens in the model organism Saccharomyces cerevisiae and is thus termed SL/DS for simplicity, we found that CQ inhibits the thiamine transporters Thi7, Nrt1, and Thi72 in yeast. We first discovered a thi3Δ mutant as hypersensitive to CQ using a genome-wide DGSL analysis. Using genome-wide GGSL and DS screens, we then found that a thi7Δ mutation confers severe growth defect in the thi3Δ mutant and that THI7 overexpression suppresses CQ-hypersensitivity of this mutant. We subsequently showed that CQ inhibits the functions of Thi7 and its homologues Nrt1 and Thi72. In particular, the transporter activity of wild-type Thi7 but not a CQ-resistant mutant (Thi7(T287N)) was completely inhibited by the drug. Similar effects were also observed with other quinoline-containing antimalarials. In addition, CQ completely inhibited a human thiamine transporter (SLC19A3) expressed in yeast and significantly inhibited thiamine uptake in cultured human cell lines. Therefore, inhibition of thiamine uptake is a conserved mechanism of action of CQ. This study also demonstrated SL/DS as a uniquely effective methodology for discovering drug targets.

Enhanced anticancer activity of gemcitabine coupling with conjugated linoleic acid against human breast cancer in vitro and in vivo.

Gemcitabine (GEM) is a nucleoside analog agent against a wide variety of tumors. To overcome its limitation of rapid metabolism in vivo that results in short circulation time and poor antitumor efficacy, a novel prodrug (CLA-GEM conjugate) has been developed through the covalent coupling of conjugated linoleic acid (CLA) to N(4)-amino group of GEM. The chemical structure of CLA-GEM conjugate was identified by NMR, FTIR and other methods. From in vitro tests, it was demonstrated that the linkage with CLA increased the plasma stability of GEM as well as the antitumor activity against human breast tumor cells (MCF-7). Importantly, it also altered the transport pattern of GEM across cell membrane (MCF-7 and MDA-MB-231), evidenced by the little effect of nucleoside transporter inhibitors (NBMPR and dipyridamole) on the IC(50) values of CLA-GEM, instead of the great effect on that of unmodified GEM. In vivo pharmacokinetic study showed that the CLA-GEM conjugate had a longer plasma half-life and a higher bioavailability compared to that of unmodified GEM. Significant stronger antitumor activity was observed in the nude mice xenografted MCF-7 breast tumor after treated with CLA-GEM than that of unmodified GEM, while no significant body weight loss was found in all treatments. In conclusion, the novel CLA-GEM conjugate prepared in this study would be a promising prodrug of gemcitabine for future clinical use.

Glucose decreases extracellular adenosine levels in isolated mouse and rat pancreatic islets.

The pancreatic islets of Langerhans are responsible for the regulated release of the endocrine hormones insulin and glucagon that participate in the control of glucose homeostasis. Abnormal regulation of these hormones can result in glucose intolerance and lead to the development of diabetes. Numerous efforts have been made to better understand the physiological regulators of insulin and glucagon secretion. One of these regulators is the purine nucleoside, adenosine. Though exogenous application of adenosine has been demonstrated to stimulate glucagon release and inhibit insulin release, the physiological significance of this pathway has been unclear. We used a novel 7 µm enzyme-coated electrode biosensor to measure adenosine levels in isolated rodent islets. In the mouse islets, basal adenosine levels in the presence of 3 mM glucose were estimated to be 5.7 ± 0.6 µM. As glucose was increased, extracellular adenosine diminished. A 10-fold increase of extracellular KCl increased adenosine levels to 16.4 ± 2.0 µM. This release required extracellular Ca (2+) suggesting that it occurred via an exocytosis-dependent mechanism. We also found that while rat islets were able to convert exogenous ATP into adenosine, mouse islets were unable to do this. Our study demonstrates for the first time the basal levels of adenosine and its inverse relationship to extracellular glucose in pancreatic islets.

Equilibrative nucleoside transporter 1 (ENT1) regulates postischemic blood flow during acute kidney injury in mice.

A complex biologic network regulates kidney perfusion under physiologic conditions. This system is profoundly perturbed following renal ischemia, a leading cause of acute kidney injury (AKI) - a life-threatening condition that frequently complicates the care of hospitalized patients. Therapeutic approaches to prevent and treat AKI are extremely limited. Better understanding of the molecular pathways promoting postischemic reflow could provide new candidate targets for AKI therapeutics. Due to its role in adapting tissues to hypoxia, we hypothesized that extracellular adenosine has a regulatory function in the postischemic control of renal perfusion. Consistent with the notion that equilibrative nucleoside transporters (ENTs) terminate adenosine signaling, we observed that pharmacologic ENT inhibition in mice elevated renal adenosine levels and dampened AKI. Deletion of the ENTs resulted in selective protection in Ent1-/- mice. Comprehensive examination of adenosine receptor-knockout mice exposed to AKI demonstrated that renal protection by ENT inhibitors involves the A2B adenosine receptor. Indeed, crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia effectively prevented a postischemic no-reflow phenomenon. These studies identify ENT1 and adenosine receptors as key to the process of reestablishing renal perfusion following ischemic AKI. If translatable from mice to humans, these data have important therapeutic implications.

Adenosine negatively regulates duodenal motility in mice: role of A(1) and A(2A) receptors.

BACKGROUND AND PURPOSE: Adenosine is considered to be an important modulator of intestinal motility. This study was undertaken to investigate the role of adenosine in the modulation of contractility in the mouse duodenum and to characterize the adenosine receptor subtypes involved. EXPERIMENTAL APPROACH: RT-PCR was used to investigate the expression of mRNA encoding for A(1), A(2A), A(2B) and A(3) receptors. Contractile activity was examined in vitro as changes in isometric tension. KEY RESULTS: In mouse duodenum, all four classes of adenosine receptors were expressed, with the A(2B) receptor subtype being confined to the mucosal layer. Adenosine caused relaxation of mouse longitudinal duodenal muscle; this was antagonized by the A(1) receptor antagonist and mimicked by N(6) -cyclopentyladenosine (CPA), selective A(1) agonist. The relaxation induced by A(1) receptor activation was insensitive to tetrodotoxin (TTX) or N(ω) -nitro-l-arginine methyl ester (l-NAME). Adenosine also inhibited cholinergic contractions evoked by neural stimulation, effect reversed by the A(1) receptor antagonist, but not myogenic contractions induced by carbachol. CPA and 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680), A(2A) receptor agonist, both inhibited the nerve-evoked cholinergic contractions. l-NAME prevented only the CGS-21680-induced effects. S-(4-Nitrobenzyl)-6-thioinosine, a nucleoside uptake inhibitor, reduced the amplitude of nerve-evoked cholinergic contractions, an effect reversed by an A(2A) receptor antagonist or l-NAME. CONCLUSIONS AND IMPLICATIONS: Adenosine can negatively regulate mouse duodenal motility either by activating A(1) inhibitory receptors located post-junctionally or controlling neurotransmitter release via A(1) or A(2A) receptors. Both receptors are available for pharmacological recruitment, even if only A(2A) receptors appear to be preferentially stimulated by endogenous adenosine.

Prevention of early loss of transplanted islets in the liver of mice by adenosine.

BACKGROUND: The low efficiency of islet transplantation necessitating sequential transplantations with the use of 2 to 3 donors for a recipient has been a major obstacle facing clinical islet transplantation. We determined whether adenosine has any beneficial effects on preventing early loss of transplanted islets in the liver, thereby facilitating successful islet transplantation from one donor to one recipient in mice. METHODS: Two hundred islets, the number of islets from a single mouse pancreas, were grafted into the liver of streptozotocin-induced diabetic C57BL/6 mice. Adenosine was administered once at the time of islet transplantation. Mononuclear cells in the liver of mice receiving islets were isolated and examined by flow cytometry. RESULTS: A single injection of adenosine at the time of transplantation ameliorated hyperglycemia of diabetic mice receiving 200 syngenic islets with suppression of interferon (IFN)-gamma production of hepatic NKT cells and neutrophils, while that of control did not. The IFN-gamma production of NKT cells and neutrophils in the liver of mice treated with alpha-galactosylceramide, a synthetic ligand of NKT cells was suppressed by adenosine. The beneficial effect of adenosine was also observed for BALB/c islet allografts when alloimmune rejection was prevented by anti-CD4 antibody. CONCLUSIONS: Adenosine suppresses the NKT cell-mediated IFN-gamma production of neutrophils in the liver of mice receiving islets, thus leading to prevention of early loss of transplanted syngenic and allogenic islets. The findings indicate that adenosine may improve efficiency of clinical islet transplantation.

Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate.

At the rat motor nerve terminals, activation of muscarinic M(1) receptors negatively modulates the activity of inhibitory muscarinic M(2) receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involved endogenous adenosine tonically activating A(1) receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [(3)H]-choline (2.5 microCi/ml). Selective activation of muscarinic M(1) and adenosine A(1) receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 microM) and R-N(6)-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [(3)H]-ACh release induced by muscarinic M(2) receptor activation with oxotremorine (10 microM). Attenuation of the inhibitory effect of oxotremorine (10 microM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M(1) autoreceptors, suggesting that suppression of M(2)-inhibiton by A(1) receptor activation is independent on muscarinic M(1) receptor activity. Conversely, the negative crosstalk between M(1) and M(2) autoreceptors seems to involve endogenous adenosine tonically activating A(1) receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 microM) by McN-A-343 (3 microM) was suppressed by the A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 microM). The results suggest that the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A(1) receptors at the rat motor endplate.

Reduced ribavirin antiviral efficacy via nucleoside transporter-mediated drug resistance.

Treatment for hepatitis C virus infection currently consists of pegylated interferon and ribavirin (RBV), a nucleoside analog. Although RBV clearly plays a role in aiding the treatment response, its antiviral mechanism is unclear. Regardless of the specific mechanism of RBV, we hypothesize that differences in levels of cellular uptake of RBV may affect antiviral efficacy and treatment success and that cells may become RBV resistant through reduced uptake. We monitored RBV uptake in various cell lines and determined the effect of uptake capacity on viral replication. RBV-resistant cells demonstrated reduced RBV uptake and increased growth of a model RNA virus, poliovirus, in the presence of RBV. Overexpression of equilibrative nucleoside transporter 1 (ENT1) or concentrative nucleoside transporter 3 (CNT3) increased RBV uptake in RBV-sensitive cell lines and restored the uptake defect in most RBV-resistant cell lines. However, CNT3 is not expressed in Huh-7 liver cells, and inhibition of concentrative transport did not affect RBV uptake. Blocking equilibrative transport using the inhibitor nitrobenzylmercaptopurine riboside recapitulated the RBV-resistant phenotype in RBV-sensitive cell lines, with a reduction in RBV uptake and increased poliovirus growth. Taken together, these results indicate that RBV uptake is restricted primarily to ENT1 in the cell lines examined. Interestingly, some RBV-resistant cell lines may compensate for reduced ENT1-mediated nucleoside uptake by increasing the activity of an alternative nucleoside transporter, ENT2. It is possible that RBV uptake affects the antiviral treatment response, either through natural differences in patients or through acquired resistance.

Adenosine as an endogenous regulating factor of hippocampal sharp waves.

The rodent hippocampus exhibits population activities called sharp waves (SPWs) during slow wave sleep and wake immobility. SPWs are important for hippocampal-cortical communication and memory consolidation, and abnormal sharp wave-ripple complexes are closely related to epileptic seizures. Although the SPWs are known to arise from the CA3 circuit, the local mechanisms underlying their generation are not fully understood. We hypothesize that endogenous adenosine is a local regulator of hippocampal SPWs. We tested this hypothesis in thick mouse hippocampal slices that encompass a relatively large hippocampal circuit and have a high propensity of generating spontaneous in vitro SPWs. We found that application of adenosine A1 receptor antagonists induced in vitro SPWs and that such induction was sensitive to blockade by NMDA receptor antagonists. By contrast, an increase in endogenous adenosine via pharmacological inhibition of adenosine transporters or adenosine degrading enzymes suppressed spontaneous in vitro SPWs. We thus suggest that the initiation and incidence of sharp wave-like population events are under tight control by the activity of endogenously stimulated A1 receptors.