Characterization of Equilibrative Nucleoside Transport of the Pancreatic Cancer Cell Line: Panc-1.

Objectives: Gemcitabine, a first-line chemotherapeutic nucleoside analog drug (NAD) for pancreatic cancer, faces limitations due to drug resistance. Characterizing pancreatic cancer cells' transport characteristics may help identify the mechanisms behind drug resistance, and develop more effective therapeutic strategies. Therefore, in this study, we aimed to determine the nucleoside transport properties of Panc-1 cells, one of the commonly used pancreatic adenocarcinoma cell lines. Materials and Methods: To assess the presence of equilibrative nucleoside transporter-1 (ENT-1) in Panc-1 cells, we performed immunofluorescence staining, western blot analysis, and S-(4-nitrobenzyl)-6-thioinosine (NBTI) binding assays. We also conducted standard uptake assays to measure the sodium-independent uptake of [3H]-labeled chloroadenosine, hypoxanthine, and uridine. In addition, we determined the half-maximal inhibitory concentration (IC50) of gemcitabine. Statistical analyses were performed using GraphPad Prism version 8.0 for Windows. Results: The sodium-independent uptake of [3H]-labeled chloroadenosine, hypoxanthine, and uridine was measured using standard uptake assays, and the transport rates were determined as 111.1 ± 3.4 pmol/mg protein/10 s, 62.5 ± 4.8 pmol/mg protein/10 s, and 101.3 ± 2.5 pmol/mg protein/10 s, respectively. Furthermore, the presence of ENT-1 protein was confirmed using NBTI binding assays (Bmax 1.52 ± 0.1 pmol/mg protein; equilibrium dissociation constant 0.42 ± 0.1 nM). Immunofluorescence assays and western blot analysis also revealed ENT-1 in Panc-1 cells. The determined IC50 of gemcitabine in Panc-1 cells was 2 µM, indicating moderate sensitivity. Conclusion: These results suggest that Panc-1 is a suitable preclinical cellular model for studying NAD transport properties and potential therapies in pancreatic cancer and pharmaceutical research.

Adenosinergic system and nucleoside transporters in attention deficit hyperactivity disorder: Current findings.

Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder with high heterogeneity that can affect individuals of any age. It is characterized by three main symptoms: inattention, hyperactivity, and impulsivity. These neurobehavioral alterations and neurochemical and pharmacological findings are mainly attributed to unbalanced catecholaminergic signaling, especially involving dopaminergic pathways within prefrontal and striatal areas. Dopamine receptors and transporters are not solely implicated in this imbalance, as evidence indicates that the dopaminergic signaling is modulated by adenosine activity. To this extent, alterations in adenosinergic signaling are probably involved in ADHD. Here, we review the current knowledge about adenosine's role in the modulation of chemical, behavioral and cognitive parameters of ADHD, especially regarding dopaminergic signaling. Current literature usually links adenosine receptors signaling to the dopaminergic imbalance found in ADHD, but there is evidence that equilibrative nucleoside transporters (ENTs) could also be implicated as players in dopaminergic signaling alterations seen in ADHD, since their involvement in other neurobehavioral impairments.

Anticonvulsant effect of equilibrative nucleoside transporters 1 inhibitor in a mouse model of Dravet syndrome.

There are limited therapeutic options for patients with Dravet syndrome (DS). The equilibrative nucleoside transporters 1 (ENT1) mediate both the influx and efflux of adenosine across the cell membrane exerted beneficial effects in the treatment of epilepsy. This study aimed to evaluate the anticonvulsant effect of the ENT1 inhibitor in an animal model of DS (Scn1aE1099X/+ mice). J7 (5 mg/kg) treatment was efficacious in elevating seizure threshold in Scn1aE1099X/+ mice after hyperthermia exposure. Moreover, the J7 treatment significantly reduced the frequency of spontaneous excitatory post-synaptic currents (sEPSCs, ~35% reduction) without affecting the amplitude in dentate gyrus (DG) granule cells. Pretreatment with the adenosine A1 receptor (A1R) antagonist, DPCPX, abolished the J7 effects on sEPSCs. These observations suggest that the J7 shows an anticonvulsant effect in hyperthermia-induced seizures in Scn1aE1099X/+ mice. This effect possibly acts on presynaptic A1R-mediated signaling modulation in granule cells.

Visualization research on ENT1/NIS dual-function gene therapy to reverse drug resistance mediated by MUC1 in GEM-resistant pancreatic cancer.

PURPOSE: To use bifunctional target genes to increase the intracellular transport of gemcitabine (GEM) to reverse chemotherapy resistance and to simultaneously use reporter gene imaging to localize therapeutic genes. The therapeutic effect was evaluated by [18F]FLT PET/CT to visualize the effect of gene therapy. METHODS: A viral gene vector containing the pancreatic cancer-targeting promoter MUC1 for specific transcription of equilibrative nucleoside transporter 1 (ENT1) and NIS (nuclide transport channel) was employed. [125I]NaI uptake tests and [131I]NaI SPECT imaging were performed to verify the function of NIS and the target function of MUC1. The correlation between [18F]FLT uptake and GEM resistance were assessed, and the influence ENT1 and thymidine kinase 1 (TK1) expression on [18F]FLT micro-PET/CT was measured, which provides a theoretical basis for the use of [18F]FLT micro-PET/CT to evaluate the efficacy of gene therapy. RESULTS: First, functions of gene therapy were confirmed: ENT1 reversed the drug resistance of GEM-resistant pancreatic cancer cells by increasing GEM intracellular transport; MUC1 drove NIS target gene expression in pancreatic cancer; and therapeutic genes could be localized using [131I]NaI SPECT reporter gene imaging. Second, the [18F]FLT uptake ratio was affected by drug resistance and GEM treatment. The mechanism underlying this effect was related to ENT1 and TK1. Increased expression of ENT1 inhibited the expression of TK1 after GEM chemotherapy to reduce the uptake of [18F]FLT. Finally, micro-PET/CT indicated that the SUVmax of [18F]FLT could predict survival time. SUVmax exhibited an increasing trend in resistant pancreatic cancer but a trend of inhibition after upregulation of ENT1, which was more significant after GEM treatment. CONCLUSIONS: Bifunctional targeted genes can localize therapeutic genes through reporter gene imaging, reverse the drug resistance of GEM-resistant pancreatic cancer and be visually evaluated through [18F]FLT micro-PET/CT.

Stiffness and axial pain are associated with the progression of calcification in a mouse model of diffuse idiopathic skeletal hyperostosis.

BACKGROUND: Diffuse idiopathic skeletal hyperostosis (DISH) is characterized by progressive calcification of spinal tissues; however, the impact of calcification on pain and function is poorly understood. This study examined the association between progressive ectopic spine calcification in mice lacking equilibrative nucleoside transporter 1 (ENT1-/-), a preclinical model of DISH, and behavioral indicators of pain. METHODS: A longitudinal study design was used to assess radiating pain, axial discomfort, and physical function in wild-type and ENT1-/- mice at 2, 4, and 6 months. At endpoint, spinal cords were isolated for immunohistochemical analysis of astrocytes (GFAP), microglia (IBA1), and nociceptive innervation (CGRP). RESULTS: Increased spine calcification in ENT1-/- mice was associated with reductions in flexmaze exploration, vertical activity in an open field, and self-supporting behavior in tail suspension, suggesting flexion-induced discomfort or stiffness. Grip force during the axial stretch was also reduced in ENT1-/- mice at 6 months of age. Increased CGRP immunoreactivity was detected in the spinal cords of female and male ENT1-/- mice compared to wild-type. GFAP- and IBA1-immunoreactivity were increased in female ENT1-/- mice compared to wild-type, suggesting an increase in nociceptive innervation. CONCLUSION: These data suggest that ENT1-/- mice experience axial discomfort and/or stiffness and importantly that these features are detected during the early stages of spine calcification.

Erythrocyte type 1 equilibrative nucleoside transporter expression in sickle cell disease and sickle cell trait.

Hypoxia-mediated red blood cell (RBC) sickling is central to the pathophysiology of sickle cell disease (SCD). The signalling nucleoside adenosine is thought to play a significant role in this process. This study investigated expression of the erythrocyte type 1 equilibrative nucleoside transporter (ENT1), a key regulator of plasma adenosine, in adult patients with SCD and carriers of sickle cell trait (SCT). Relative quantitative expression analysis of erythrocyte ENT1 was carried out by Western blot and flow cytometry. Patients with SCD with steady state conditions, either with SS or SC genotype, untreated or under hydroxycarbamide (HC) treatment, exhibited a relatively high variability of erythrocyte ENT1, but with levels not significantly different from normal controls. Most strikingly, expression of erythrocyte ENT1 was found to be significantly decreased in patients with SCD undergoing painful vaso-occlusive episode and, unexpectedly, also in healthy SCT carriers. Promoting hypoxia-induced adenosine signalling, the reduced expression of erythrocyte ENT1 might contribute to the pathophysiology of SCD and to the susceptibility of SCT individuals to altitude hypoxia or exercise to exhaustion.

Adenosine derivatives from Cordyceps exert antitumor effects against ovarian cancer cells through ENT1-mediated transport, induction of AMPK signaling, and consequent autophagic cell death.

Cordyceps militaris is rich in adenosine derivatives, including 3'-deoxyadenosine, also known as cordycepin. It has been reported for antitumor effects, but its underlying molecular mechanism has yet to be elucidated. We investigated how adenosine derivatives exerted antitumor effects against ovarian cancer using human ovarian cancer cells and a xenograft mouse model. Treatment with adenosine derivatives effectively resulted in cell death of ovarian cancer cells through AMPK activation and subsequently mTOR-mediated autophagic induction. Intriguingly, the effect required membrane transport of adenosine derivatives via ENT1, rather than ADORA-mediated cellular signaling. Our data suggest that adenosine derivatives may be an effective therapeutic intervention in ovarian cancer through induction of ENT1-AMPK-mTOR-mediated autophagic cell death.

A purinergic call to arms from dying brown adipocytes.

Brown adipocytes react to temperature and nutritional challenges by ramping up their metabolism and generating heat. This adaptation to changes in the environment is crucial for defending organismal homeostasis, but is impaired in obesity and during aging. Writing in Nature, Niemann et al. show that brown adipocytes become apoptotic under thermoneutral conditions and release ATP, which in turn is converted extracellularly into inosine. They further present evidence that pharmacological and genetic manipulations that enhance signalling of this purine metabolite stimulates thermogenesis in brown adipocytes and promotes metabolic health.

The Hypoxia-Adenosine Link during Myocardial Ischemia-Reperfusion Injury.

Despite increasing availability and more successful interventional approaches to restore coronary reperfusion, myocardial ischemia-reperfusion injury is a substantial cause of morbidity and mortality worldwide. During myocardial ischemia, the myocardium becomes profoundly hypoxic, thus causing stabilization of hypoxia-inducible transcription factors (HIF). Stabilization of HIF leads to a transcriptional program that promotes adaptation to hypoxia and cellular survival. Transcriptional consequences of HIF stabilization include increases in extracellular production and signaling effects of adenosine. Extracellular adenosine functions as a signaling molecule via the activation of adenosine receptors. Several studies implicated adenosine signaling in cardioprotection, particularly through the activation of the Adora2a and Adora2b receptors. Adenosine receptor activation can lead to metabolic adaptation to enhance ischemia tolerance or dampen myocardial reperfusion injury via signaling events on immune cells. Many studies highlight that clinical strategies to target the hypoxia-adenosine link could be considered for clinical trials. This could be achieved by using pharmacologic HIF activators or by directly enhancing extracellular adenosine production or signaling as a therapy for patients with acute myocardial infarction, or undergoing cardiac surgery.

Augustine Blood Group System and Equilibrative Nucleoside Transporter 1.

Augustine (AUG) is a blood group system comprising four antigens: AUG1, AUG2 (Ata), and AUG4 are of very high frequency; AUG3 is of very low frequency. These antigens are located on ENT1, an equilibrative nucleoside transporter encoded by SLC19A1. AUG antibodies are of clinical relevance in blood transfusion and pregnancy: anti-AUG2 have caused haemolytic transfusion reactions; the only anti-AUG3 was associated with severe haemolytic disease of the fetus and newborn. ENT1 is present in almost all human tissues. It facilitates the transfer of purine and pyrimidine nucleosides and is responsible for the majority of adenosine transport across plasma membranes. Adenosine transport appears to be an important factor in the regulation of bone metabolism. The AUGnull phenotype (AUG:-1,-2,-3,-4) has been found in three siblings, who are homozygous for an inactivating splice-site mutation in SLC29A1. Although ENT1 is very likely to be absent from all cells in these three individuals, they were apparently healthy with normal lifestyles. However, they suffered frequent attacks of pseudogout, a form of arthritis, in various joints with multiple calcifications around their hand joints. Ectopic calcification in the hips, pubic symphysis, and lumbar discs was present in the propositus. The three AUGnull individuals had misshapen red cells with deregulated protein phosphorylation, but no anaemia or shortening of red cell lifespan. Defective in vitro erythropoiesis in the absence of ENT1 was confirmed by shRNA-mediated knockdown of ENT1 during in vitro erythropoiesis of CD34+ progenitor cells from individuals with normal ENT1. Nucleoside transporters, such as ENT1, are vital in the uptake of synthetic nucleoside analogue drugs, used in cancer and viral chemotherapy. It is feasible that the efficacy of these drugs would be compromised in patients with the extremely rare AUGnull phenotype.

A review of the effects of ticagrelor on adenosine concentration and its clinical significance.

BACKGROUND: Ticagrelor is an oral antiplatelet drug that can reversibly bind to the platelet P2Y12 receptor. Ticagrelor is metabolized mainly by CYP3A4 and produces a rapid blood concentration-dependent platelet inhibitory effect. Unlike other P2Y12 receptor antagonists, many clinical features of ticagrelor are not related to P2Y12 receptor antagonism. PURPOSE: This review aims to gather existing literature on the clinical effects of ticagrelor after inhibiting adenosine uptake. METHODOLOGY: The current study reviewed literature related to the effects of ticagrelor on adenosine metabolism. The review also examined the drug's biological effects and clinical characteristics to see how it could be used in a clinical setting. RESULTS: Many studies have shown that ticagrelor can inhibit equilibrative nucleoside transporter 1 (ENT1). This inhibition leads to intracellular adenosine uptake, increased adenosine half-life and plasma concentration levels and an enhanced adenosine-mediated biological effect. CONCLUSIONS: Based on the studies reviewed, it was found that ticagrelor essentially inhibits adenosine absorption of adenosine into cells through ENT1, which increases the concentration in the blood and subsequently increases the protection of the heart muscle by adenosine. It also prevents platelet aggregation, and extends the biological effects of coronary arteries. Moreover, it leads to a lower mortality rate in acute coronary syndrome (ACS) patients.

The Longer the Block, the Harder You Fall: Extrinsic Idiopathic Atrioventricular Block Masquerading as Seizures.

History and physical examination are the diagnostic cornerstones of transient loss of consciousness (TLOC). However, details can be scarce and examination unrevealing, thus making the diagnosis elusive. In a case of convulsive TLOC, the initial diagnosis was incorrect, but a fortuitously captured event on telemetry yielded the diagnosis: extrinsic idiopathic atrioventricular block. (Level of Difficulty: Beginner.).

Equilibrative nucleoside transporter 1 inhibition rescues energy dysfunction and pathology in a model of tauopathy.

Tau pathology is instrumental in the gradual loss of neuronal functions and cognitive decline in tauopathies, including Alzheimer's disease (AD). Earlier reports showed that adenosine metabolism is abnormal in the brain of AD patients while consequences remained ill-defined. Herein, we aimed at investigating whether manipulation of adenosine tone would impact Tau pathology, associated molecular alterations and subsequent neurodegeneration. We demonstrated that treatment with an inhibitor (J4) of equilibrative nucleoside transporter 1 (ENT1) exerted beneficial effects in a mouse model of Tauopathy. Treatment with J4 not only reduced Tau hyperphosphorylation but also rescued memory deficits, mitochondrial dysfunction, synaptic loss, and abnormal expression of immune-related gene signatures. These beneficial effects were particularly ascribed to the ability of J4 to suppress the overactivation of AMPK (an energy reduction sensor), suggesting that normalization of energy dysfunction mitigates neuronal dysfunctions in Tauopathy. Collectively, these data highlight that targeting adenosine metabolism is a novel strategy for tauopathies.

Adaptative mechanism of the equilibrative nucleoside transporter 1 (ENT-1) and blood adenosine levels in elite freedivers.

PURPOSE: Long static or intense dynamic apnoea-like high-altitude exposure is inducing hypoxia. Adenosine is known to participate to the adaptive response to hypoxia leading to the control of heart rate, blood pressure and vasodilation. Extracellular adenosine level is controlled through the equilibrative nucleoside transporter 1 (ENT-1) and the enzyme adenosine deaminase (ADA). The aim of this study was to determine the control of adenosine blood level (ABL) via ENT-1 and ADA during apnoea-induced hypoxia in elite freedivers was similar to high-altitude adaptation. METHODS: Ten freediver champions and ten controls were studied. Biological (e.g. ENT-1, ADA, ABL, PaO2, PaCO2 and pH) and cardiovascular (e.g. heart rate, arterial pressure) parameters were measured at rest and after a submaximal dry static apnoea. RESULTS: In freedivers, ABL was higher than in control participants in basal condition and increased more in response to apnoea. Also, freedivers showed an ADA increased in response to apnoea. Finally, ENT-1 level and function were reduced for the free divers. CONCLUSION: Our results suggest in freedivers the presence of an adaptive mechanism similar to the one observed in human exposed to chronic hypoxia induced by high-altitude environment.

Dynamic-related protein 1 inhibitor eases epileptic seizures and can regulate equilibrative nucleoside transporter 1 expression.

BACKGROUND: Dynamic-related protein 1 (Drp1) is a key protein involved in the regulation of mitochondrial fission, and it could affect the dynamic balance of mitochondria and appears to be protective against neuronal injury in epileptic seizures. Equilibrative nucleoside transporter 1 (ENT1) is expressed and functional in the mitochondrial membrane that equilibrates adenosine concentration across membranes. Whether Drp1 participates in the pathogenesis of epileptic seizures via regulating function of ENT1 remains unclear. METHODS: In the present study, we used pilocarpine to induce status epilepticus (SE) in rats, and we used mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor to Drp1, to suppress mitochondrial fission in pilocarpine-induced SE model. Mdivi-1administered by intraperitoneal injection before SE induction, and the latency to firstepileptic seizure and the number of epileptic seizures was thereafter observed. The distribution of Drp1 was detected by immunofluorescence, and the expression patterns of Drp1 and ENT1 were detected by Western blot. Furthermore, the mitochondrial ultrastructure of neurons in the hippocampal CA1 region was observed by transmission electron microscopy. RESULTS: We found that Drp1 was expressed mainly in neurons and Drp1 expression was significantly upregulated in the hippocampal and temporal neocortex tissues at 6 h and 24 h after induction of SE. Mitochondrial fission inhibitor 1 attenuated epileptic seizures after induction of SE, reduced mitochondrial damage and ENT1 expression. CONCLUSIONS: These data indicate that Drp1 is upregulated in hippocampus and temporal neocortex after pilocarpine-induced SE and the inhibition of Drp1 may lead to potential therapeutic target for SE by regulating ENT1 after pilocarpine-induced SE.

Decreased Equilibrative Nucleoside Transporter 1 (ENT1) Activity Contributes to the High Extracellular Adenosine Levels in Mesenchymal Glioblastoma Stem-Like Cells.

Glioblastoma multiforme is one of the most malignant types of cancer. This is mainly due to a cell subpopulation with an extremely aggressive potential, called glioblastoma stem-like cells (GSCs). These cells produce high levels of extracellular adenosine which has been associated with increased chemoresistance, migration, and invasion in glioblastoma. In this study, we attempted to elucidate the mechanisms that control extracellular adenosine levels in GSC subtypes. By using primary and U87MG-derived GSCs, we associated increased extracellular adenosine with the mesenchymal phenotype. [3H]-adenosine uptake occurred mainly through the equilibrative nucleoside transporters (ENTs) in GSCs, but mesenchymal GSCs have lower expression and ENT1-mediated uptake activity than proneural GSCs. By analyzing expression and enzymatic activity, we determined that ecto-5'-nucleotidase (CD73) is predominantly expressed in proneural GSCs, driving AMPase activity. While in mesenchymal GSCs, both CD73 and Prostatic Acid Phosphatase (PAP) contribute to the AMP (adenosine monophosphate) hydrolysis. We did not observe significant differences between the expression of proteins involved in the metabolization of adenosine among the GCSs subtypes. In conclusion, the lower expression and activity of the ENT1 transporter in mesenchymal GSCs contributes to the high level of extracellular adenosine that these GSCs present.

Sustained adenosine exposure causes endothelial mitochondrial dysfunction via equilibrative nucleoside transporters.

Adenosine is a potent signaling molecule that has paradoxical effects on lung diseases. We have previously demonstrated that sustained adenosine exposure by inhibition of adenosine degradation impairs lung endothelial barrier integrity and causes intrinsic apoptosis through equilibrative nucleoside transporter1/2-mediated intracellular adenosine signaling. In this study, we further demonstrated that sustained adenosine exposure increased mitochondrial reactive oxygen species and reduced mitochondrial respiration via equilibrative nucleoside transporter1/2, but not via adenosine receptor-mediated signaling. We have previously shown that sustained adenosine exposure activates p38 and c-Jun N-terminal kinases in mitochondria. Here, we show that activation of p38 and JNK partially contributed to sustained adenosine-induced mitochondrial reactive oxygen species production. We also found that sustained adenosine exposure promoted mitochondrial fission and increased mitophagy. Finally, mitochondria-targeted antioxidants prevented sustained adenosine exposure-induced mitochondrial fission and improved cell survival. Our results suggest that inhibition of equilibrative nucleoside transporter1/2 and mitochondria-targeted antioxidants may be potential therapeutic approaches for lung diseases associated with sustained elevated adenosine.

ENT1 inhibition attenuates apoptosis by activation of cAMP/pCREB/Bcl2 pathway after MCAO in rats.

BACKGROUND AND PURPOSE: The present study was designed to investigate the potential role and the mechanism of equilibrative nucleoside transporter 1 (ENT1) on neuronal apoptosis and neurological deficits after middle cerebral artery occlusion (MCAO) in rats. METHODS: One hundred and thirty-four male Sprague-Dawley rats were subjected to two hours of MCAO followed by reperfusion. The time course of the expression level of ENT1 and phosphorylation of CREB were detected by western blot and immunofluorescence staining. Another set of animals were administrated with NBTI, the ENT1 inhibitor, by daily intraperitoneal injection starting at 0.5 h post-MCAO, infarction volume and neurological deficits were measured both at 24 h and 72 h post MCAO. We further explored the neuroprotection machenism by using H89, cAMP dependent protein kinase inhibitor, the expression of Bcl-2, Bax, phosphorylated CREB and Cleaved caspase-3 were quantified by Western blot, neuronal apoptosis were analyed by TUNEL staining. RESULTS: The endogenous expression of ENT1 were significantly increased and peaked at 12 h after MCAO. High-dose of NBTI (15 mg/kg) reduced brain infarction volume and improved neurologic deficits both at 24 h and 72 h post MCAO. Moreover, NBTI significantly increased the level of CREB phosphorylation and extracellular adenosine concentration, and decreased the neuronal apoptosis 24 h after MCAO. NBTI treatment reduced the expression of Bax and cleaved caspase-3, while up-regulated Bcl-2 compared with vehicle group. These effects were abolished by H89 pretreatment. CONCLUSIONS: ENT1 inhibition prevented neuronal apoptosis and improves neurological deficits through cAMP/PKA/CREB/Bcl-2 signaling pathway after MCAO in rats. ENT1 might be an effective target in the treatment strategy for ischemic stroke.

The failure of DAC to induce OCT2 expression and its remission by hemoglobin-based nanocarriers under hypoxia in renal cell carcinoma.

Background: Human organic cation transporter 2 (OCT2) is the most abundant and important uptake transporter involved in the renal excretion of cationic drugs. Abnormal hypermethylation- mediated silencing of OCT2 results in oxaliplatin resistance in renal cell carcinoma (RCC). The epigenetic activation of OCT2 by decitabine (DAC) reversed this resistance in normoxic conditions. Given the hypoxic characteristic of RCC, it is still unclear whether hypoxia promotes DAC resistance and is involved in the regulation of OCT2. Methods: The mRNA and protein expression of OCT2 was determined by qRT-PCR and Western blotting. MSRE-qPCR and BSP were used to examine methylation modifications at the OCT2 promoter. The ChIP-qPCR analysis was performed to detect the abundance of histone modification and HIF-1α. The accumulation of DAC and 5-mC were detected using LC-MS, and the amount of 5-hmC was determined by dot blot analysis. To understand the role of hypoxia in the regulation of equilibrative nucleoside transporter 1 (ENT1) expression, the HIF-1α KO cell model was constructed. The re-emulsion method was used for the construction of H-NPs, an oxygen nanocarrier based on hemoglobin, to alleviate the drug resistance of DAC under hypoxia. Results: DAC was unable to upregulate OCT2 expression in hypoxic conditions because of the hypermethylation and low H3K4me3 modification in its promoter region. Hypoxia-mediated repression of human ENT1, which was markedly suppressed in RCC, resulted in a decrease in the cellular accumulation of DAC. Besides, hypoxia-induced upregulation of histone deacetylase HDAC9, which impaired the enrichment of H3K27ac modification in the OCT2 promoter, led to the transcriptional repression of OCT2. H-NPs could attenuate the hypoxia-induced loss of DAC activity and sensitize RCC cells to the sequential combination therapy of DAC and oxaliplatin. Conclusions: Hypoxia-mediated repression of ENT1 led to the inability of DAC to upregulate the expression of OCT2 under hypoxia. H-NPs could alleviate resistance to oxaliplatin and DAC in RCC cells under hypoxia and may have potential clinical applications.

Nicotinic α7 receptor-induced adenosine release from perisynaptic Schwann cells controls acetylcholine spillover from motor endplates.

Acetylcholine (ACh) spillover from motor endplates occurs after neuronal firing bursts being potentiated by cholinesterase inhibitors (e.g., neostigmine). Nicotinic α7 receptors (α7nAChR) on perisynaptic Schwann cells (PSCs) can control ACh spillover by unknown mechanisms. We hypothesized that adenosine might be the gliotransmitter underlying PSCs-nerve terminal communication. Rat isolated hemidiaphragm preparations were used to measure (1) the outflow of [3 H]ACh, (2) real-time transmitter exocytosis by video-microscopy with the FM4-64 fluorescent dye, and (3) skeletal muscle contractions during high-frequency (50 Hz) nerve stimulation bursts in the presence of a selective α7nAChR agonist, PNU 282987, or upon inhibition of cholinesterase activity with neostigmine. To confirm our prediction that α7nAChR-mediated effects require direct activation of PSCs, we used fluorescence video-microscopy in the real-time mode to measure PNU 282987-induced [Ca2+ ]i transients from Fluo-4 NW loaded PSCs in non-stimulated preparations. The α7nAChR agonist, PNU 282987, decreased nerve-evoked diaphragm tetanic contractions. PNU 282987-induced inhibition was mimicked by neostigmine and results from the reduction of ACh exocytosis measured as decreases in [3 H]ACh release and FM4-64 fluorescent dye unloading. Methyllycaconitine blockage of α7nAChR and the fluoroacetate gliotoxin both prevented inhibition of nerve-evoked ACh release and PSCs [Ca2+ ]i transients triggered by PNU 282987 and neostigmine. Adenosine deamination, inhibition of the ENT1 nucleoside outflow, and blockage of A1 receptors prevented PNU 282987-induced inhibition of transmitter release. Data suggest that α7nAChR controls tetanic-induced ACh spillover from the neuromuscular synapse by promoting adenosine outflow from PSCs via ENT1 transporters and retrograde activation of presynaptic A1 inhibitory receptors.