Adenosine Kinase Inhibitor Research Articles

Inhibition of adenosine kinase attenuates inflammation and neurotoxicity in traumatic optic neuropathy

Traumatic optic neuropathy (TON) is associated with apoptosis of retinal ganglion cells. Local productions of reactive oxygen species and inflammatory mediators from activated microglial cells have been hypothesized to underlie apoptotic processes. We previously demonstrated that the anti-inflammatory effect of adenosine, through A2A receptor activation had profound protective influence against retinal injury in traumatic optic neuropathy. This protective effect is limited due to rapid cellular re-uptake of adenosine by equilibrative nucleotside transporter-1 (ENT1) or break down by adenosine kinase (AK), the key enzyme in adenosine clearance pathway. Further, the use of adenosine receptors agonists are limited by systemic side effects. Therefore, we seek to investigate the potential role of amplifying the endogenous ambient level of adenosine by pharmacological inhibition of AK. We tested our hypothesis by comparing TON-induced retinal injury in mice with and without ABT-702 treatment, a selective AK inhibitor (AKI). The retinal-protective effect of ABT-702 was demonstrated by significant reduction of Iba-1, ENT1, TNF-α, IL-6, and iNOS/nNOS protein or mRNA expression in TON as revealed by western blot and real time PCR. TON-induced superoxide anion generation and nitrotyrosine expression were reduced in ABT-702 treated mice retinal sections as determined by immunoflourescence. In addition, ABT-702 attenuated p-ERK1/2 and p-P38 activation in LPS induced activated mouse microglia cells. The results of the present investigation suggested that ABT-702 had a protective role against marked TON-induced retinal inflammation and damage by augmenting the endogenous therapeutic effects of site- and event-specific accumulation of extracellular adenosine.

Structural basis for inhibition of mycobacterial and human adenosine kinase by 7-substituted 7-(Het)aryl-7-deazaadenine ribonucleosides.

Adenosine kinase (ADK) from Mycobacterium tuberculosis (Mtb) was selected as a target for design of antimycobacterial nucleosides. Screening of 7-(het)aryl-7-deazaadenine ribonucleosides with Mtb and human (h) ADKs and testing with wild-type and drug-resistant Mtb strains identified specific inhibitors of Mtb ADK with micromolar antimycobacterial activity and low cytotoxicity. X-ray structures of complexes of Mtb and hADKs with 7-ethynyl-7-deazaadenosine showed differences in inhibitor interactions in the adenosine binding sites. 1D (1)H STD NMR experiments revealed that these inhibitors are readily accommodated into the ATP and adenosine binding sites of Mtb ADK, whereas they bind preferentially into the adenosine site of hADK. Occupation of the Mtb ADK ATP site with inhibitors and formation of catalytically less competent semiopen conformation of MtbADK after inhibitor binding in the adenosine site explain the lack of phosphorylation of 7-substituted-7-deazaadenosines. Semiempirical quantum mechanical analysis confirmed different affinity of nucleosides for the Mtb ADK adenosine and ATP sites.

Abstract 541: Adenosine Inhibits C-Kit+ Progenitor Smooth Muscle Cell (P-SMC) Infiltration In Injury-Induced Neointima Formation And Human P-SMC Growth: Role of A2B Adenosine Receptor-Mediated Growth Regulation

Adenosine inhibits injury-induced neointimal formation; however, the mechanism is incompletely understood. Marrow-derived C-Kit+ progenitor cells (P-SMCs) express a smooth muscle phenotype and contribute to injury-induced neointimal thickening. The goal of our investigation was to determine whether adenosine attenuates neointimal hyperplasia in part by inhibiting the growth of P-SMCs and to clarify which adenosine receptor subtypes are involved. Digitized pixel analysis demonstrated that administration of adenosine (20 μM) peri-arterially for 7 days in a slow-release formulation inhibited injury-induced neointimal hyperplasia in the rat carotid artery by 71% (p<0.05: neointimal thickness: 37,424 ± 18,371 pixels in vehicle-treated [n=7] versus 10,352 ± 2,824 pixels in adenosine-treated [n=7]). Microscopic analysis following immunostaining for c-Kit+ cells showed a marked decrease (> 50%) in staining for c-Kit+ cells in the neointima of rats receiving adenosine. In cultured human P-SMCs, serum-induced growth (as determined by quantification of DNA synthesis and cell number), cell-migration, and collagen synthesis was inhibited by adenosine and 2-chloroadenosine, but not by N 6 -cyclopentyladenosine (selective A 1 -receptor agonist), CGS21680 (selective A 2A -receptor agonist) or N 6 -(3-iodobenzyl)adenosine-5'-N-methyluronamide (selective A 3 -receptor agonist). This agonist profile was consistent with an A 2B -receptor-mediated effect. Augmentation of endogenous adenosine levels with erythro -9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor) and 5-iodotubericidin (adenosine kinase inhibitor) attenuated P-SMC growth. Moreover the antimitogenic effects of both exogenous and endogenous adenosine were abrogated by blockade of A 2B receptors (MRS 1754), but not by antagonism of A 1 (8-cyclopentyl-1,3-dipropylxanthine), A 2A (SCH 58261) or A 3 (VUF 5574) receptors. We conclude that adenosine, via activation of A 2B receptors, inhibits neointimal formation in part by inhibiting infiltration and growth of C-Kit+ P-SMCs. These findings suggest that A 2B receptor stimulation may prevent vascular remodeling associated with coronary artery disease, hypertension, atherosclerosis and restenosis.

Adenosine kinase inhibition protects the kidney against streptozotocin-induced diabetes through anti-inflammatory and anti-oxidant mechanisms

Adenosine provides anti-inflammatory effects in cardiovascular disease via the activation of adenosine A2A receptors; however, the physiological effect of adenosine could be limited due to its phosphorylation by adenosine kinase. We hypothesized that inhibition of adenosine kinase exacerbates extracellular adenosine levels to reduce renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in male C57BL/6 mice by daily injection of streptozotocin (50mg/kg/day, i.p. for 5 days). Control and diabetic mice were then treated with the adenosine kinase inhibitor ABT702 (1.5mg/kg, i.p. two times a week for 8 weeks, n=7–8/group) or the vehicle (5% DMSO). ABT702 treatment reduced blood glucose level in diabetic mice (∼20%; P<0.05). ABT702 also reduced albuminuria and markers of glomerular injury, nephrinuria and podocalyxin excretion levels, in diabetic mice. Renal NADPH oxidase activity and urinary thiobarbituric acid reactive substances (TBARS) excretion, indices of oxidative stress, were also elevated in diabetic mice and ABT702 significantly reduced these changes. ABT702 increased renal endothelial nitric oxide synthase expression (eNOS) and nitrate/nitrite excretion levels in diabetic mice. In addition, the diabetic mice displayed an increase in renal macrophage infiltration, in association with increased renal NFκB activation. Importantly, treatment with ABT702 significantly reduced all these inflammatory parameters (P<0.05). Furthermore, ABT702 decreased glomerular permeability and inflammation and restored the decrease in glomerular occludin expression in vitro in high glucose treated human glomerular endothelial cells. Collectively, the results suggest that the reno-protective effects of ABT702 could be attributed to the reduction in renal inflammation and oxidative stress in diabetic mice.

Uveal melanoma cell growth is inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP-dependent kinase.

To evaluate the effects and mechanism of aminoimidazole carboxamide ribonucleotide (AICAR), an AMP-dependent kinase (AMPK) activator, on the growth of uveal melanoma cell lines. Four different cell lines were treated with AICAR (1-4 mM). Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Cell cycle analysis was conducted by flow cytometry; additionally, expression of cell-cycle control proteins, cell growth transcription factors, and downstream effectors of AMPK were determined by RT-PCR and Western blot. Aminoimidazole carboxamide ribonucleotide inhibited cell growth, induced S-phase arrest, and led to AMPK activation. Aminoimidazole carboxamide ribonucleotide treatment was associated with inhibition of eukaryotic translation initiation factor 4E-BP1 phosphorylation, a marker of mammalian target of rapamycin (mTOR) pathway activity. Aminoimidazole carboxamide ribonucleotide treatment was also associated with downregulation of cyclins A and D, but had minimal effects on the phosphorylation of ribosomal protein S6 or levels of the macroautophagy marker LC3B. The effects of AICAR were abolished by treatment with dipyridamole, an adenosine transporter inhibitor that blocks the entry of AICAR into cells. Treatment with adenosine kinase inhibitor 5-iodotubericidin, which inhibits the conversion of AICAR to its 5'-phosphorylated ribotide 5-aminoimidazole-4-carboxamide-1-D-ribofuranosyl-5'-monophosphate (ZMP; the direct activator of AMPK), reversed most of the growth-inhibitory effects, indicating that some of AICAR's antiproliferative effects are mediated at least partially through AMPK activation. Aminoimidazole carboxamide ribonucleotide inhibited uveal melanoma cell proliferation partially through activation of the AMPK pathway and downregulation of cyclins A1 and D1.

Adenosine kinase as a therapeutic target in traumatic optic neuropathy

Background The purpose of this study is to understand the mechanism of traumatic optic neuropathy (TON) in order to prevent vision loss. Following traumatic insults to the optic nerve, retinal microglia cells are activated through MAP Kinase pathways and increased cytotoxic activity that causes retinal ganglion cell death [1]. Under stress condition, extracellular concentration of adenosine is likely to increase and activates an anti-inflammatory pathway through A2A adenosine receptor. But in TON, the accumulated extracellular adenosine is then transported intracellular through equilibrative nucleoside transporters (ENTs) which further gets converted into AMP by Adenosine kinase (AK), which results in low extracellular adenosine concentration. We have demonstrated that microglia activation and TNF-a release was inhibited by AK inhibitor. Based on these findings, we hypothesize that an imbalance in adenosine formation and metabolism in the retinal microglia participated by AK may contribute significantly to retinal complications in the setting of TON.

The Antiepileptic Potential of Nucleosides

Despite newly developed antiepileptic drugs to suppress epileptic symptoms, approximately one third of patients remain drug refractory. Consequently, there is an urgent need to develop more effective therapeutic approaches to treat epilepsy. A great deal of evidence suggests that endogenous nucleosides, such as adenosine (Ado), guanosine (Guo), inosine (Ino) and uridine (Urd), participate in the regulation of pathomechanisms of epilepsy. Adenosine and its analogues, together with non-adenosine (non-Ado) nucleosides (e.g., Guo, Ino and Urd), have shown antiseizure activity. Adenosine kinase (ADK) inhibitors, Ado uptake inhibitors and Ado-releasing implants also have beneficial effects on epileptic seizures. These results suggest that nucleosides and their analogues, in addition to other modulators of the nucleoside system, could provide a new opportunity for the treatment of different types of epilepsies. Therefore, the aim of this review article is to summarize our present knowledge about the nucleoside system as a promising target in the treatment of epilepsy.

Use of 11C-MPDX and PET to Study Adenosine A1 Receptor Occupancy by Nonradioactive Agonists and Antagonists

Adenosine A1 receptors (A1Rs) in human and rodent brains can be visualized with the radioligand 8-dicyclopropylmethyl-1-(11)C-methyl-3-propylxanthine ((11)C-MPDX) and PET. Here we investigated whether A1R occupancy by nonradioactive agonists and antagonists can be assessed with this technique. Small-animal PET scans with arterial blood sampling were obtained for 4 groups of isoflurane-anesthetized Wistar rats: controls (n = 7); pretreated with a centrally active A1R agonist, N(6)-cyclopentyladenosine (CPA; 0.25 mg/kg intraperitoneally; dissociation constant, 0.48 nM; n = 7); pretreated with a moderate dose of caffeine (antagonist for A1Rs and adenosine A2A receptors; 4 mg/kg intraperitoneally; dissociation constant, 11 μM; n = 6); and pretreated with a high dose of caffeine (40 mg/kg intraperitoneally; n = 6). The administration of CPA resulted in a strong reduction (>50%) in the heart rate, and caffeine administration resulted in a small increase (10%-15%). A caffeine dose of 4 mg/kg (n = 6) resulted in 65.9% A1R occupancy, and a dose of 40 mg/kg (n = 6) resulted in 98.5% occupancy (calculated from a modified Lassen plot). However, the administration of CPA resulted in an increase in (11)C-MPDX binding in the brain. Small-animal PET with (11)C-MPDX can be used to assess antagonist but not agonist binding at A1Rs. Changes in tracer uptake after the administration of CPA resembled previously reported changes induced by treatment of rats with ethanol and an adenosine kinase inhibitor (ABT702). Thus, the administration of an exogenous agonist or increasing the level of an endogenous agonist have similar effects. Agonists and antagonists may bind to different sites on the A1R protein having allosteric interactions.

Antiangiogenic effects of N6‐isopentenyladenosine, an endogenous isoprenoid end product, mediated by AMPK activation

N6-isopentenyladenosine (iPA), an end product of the mevalonate pathway with an isopentenyl chain, is already known to exert a suppressor effect against various tumors. In this work, we investigated whether iPA also directly interferes with the angiogenic process, which is fundamental to tumor growth and progression. To this end, using human umbilical vein endothelial cells (HUVECs) as a suitable in vitro model of angiogenesis, we evaluated their viability, proliferation, migration, invasion, tube formation in response to iPA, and molecular mechanisms involved. Data were corroborated in mice by using a gel plug assay. iPA dose- and time-dependently inhibited all the neoangiogenesis stages, with an IC50 of 0.98 μM. We demonstrated for the first time, by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS), that iPA was monophosphorylated into 5'-iPA-monophosphate (iPAMP) by the adenosine kinase (ADK) inside the cells. iPAMP is the active form that inhibits angiogenesis through the direct activation of AMP-kinase (AMPK). Indeed, all effects were completely reversed by pretreatment with 5-iodotubercidin (5-Itu), an ADK inhibitor. The isoprenoid intermediate isopentenyl pyrophosphate (IPP), which shares the isopentenyl moiety with iPA, was ineffective in the inhibition of angiogenesis, thus showing that the iPA structure is specific for the observed effects. In conclusion, iPA is a novel AMPK activator and could represent a useful tool for the treatment of diseases where excessive neoangiogenesis is the underlying pathology.

Role of adenosine in status epilepticus: A potential new target?

The homeostatic bioenergetic network regulator adenosine is an endogenous anticonvulsant of the brain that plays critical roles in seizure termination and postictal refractoriness. Adenosine homeostasis in the adult brain is largely under the control of metabolic clearance through adenosine kinase (ADK), expressed predominantly in astrocytes. The role of adenosine in status epilepticus (SE) appears to be a double-edged sword. We demonstrated that the severity of an SE clearly depends on the expression levels of ADK. A genetic knockdown of ADK prevented SE in a mouse model, whereas transgenic overexpression of the enzyme aggravated the SE. Therefore, ADK inhibition or adenosine augmentation might be a therapeutic strategy to terminate or attenuate an SE. On the other hand, SE triggers a surge of endogenous adenosine, which may initiate secondary events leading to epileptogenesis. Two new findings point into this direction: (1) Elevated adenosine triggers changes in the epigenome; and (2) SE triggers transient changes in ADK expression, which have been linked to neurogenesis. Although the ADK/adenosine system is an attractive target for the attenuation of an SE, the same system may also trigger downstream events related to epileptogenesis.

Adenosine kinase is a key determinant for the anti-HCV activity of ribavirin

Ribavirin (RBV) is often used in conjunction with interferon-based therapy for patients with chronic hepatitis C. There is a drastic difference in the anti-hepatitis C virus (HCV) activity of RBV between the HuH-7-derived assay system, OR6, possessing the RBV-resistant phenotype (50% effective concentration [EC50 ]: >100 µM) and the recently discovered Li23-derived assay system, ORL8, possessing the RBV-sensitive phenotype (EC50 : 8 µM; clinically achievable concentration). This is because the anti-HCV activity of RBV was mediated by the inhibition of inosine monophosphate dehydrogenase in RBV-sensitive ORL8 cells harboring HCV RNA. By means of comparative analyses using RBV-resistant OR6 cells and RBV-sensitive ORL8 cells, we tried to identify host factor(s) determining the anti-HCV activity of RBV. We found that the expression of adenosine kinase (ADK) in ORL8 cells was significantly higher than that in RBV-resistant OR6 cells harboring HCV RNA. Ectopic ADK expression in OR6 cells converted them from an RBV-resistant to an RBV-sensitive phenotype, and inhibition of ADK abolished the activity of RBV. We showed that the differential ADK expression between ORL8 and OR6 cells was not the result of genetic polymorphisms in the ADK gene promoter region and was not mediated by a microRNA control mechanism. We found that the 5' untranslated region (UTR) of ADK messenger RNA in ORL8 cells was longer than that in OR6 cells, and that only a long 5' UTR possessed internal ribosome entry site (IRES) activity. Finally, we demonstrated that the long 5' UTR functioned as an IRES in primary human hepatocytes. These results indicate that ADK acts as a determinant for the activity of RBV and provide new insight into the molecular mechanism underlying differential drug sensitivity.

ABT-702, an adenosine kinase inhibitor, attenuates inflammation in diabetic retinopathy

AimsThis study was undertaken to determine the effect of an adenosine kinase inhibitor (AKI) in diabetic retinopathy (DR). We have shown previously that adenosine signaling via A2A receptors (A2AAR) is involved in retinal protection from diabetes-induced inflammation. Here we demonstrate that AKI-enhanced adenosine signaling provides protection from DR in mice. Main methodsWe targeted AK, the key enzyme in adenosine metabolism, using a treatment regime with the selective AKI, ABT-702 (1.5mg/kg intraperitoneally twice a week) commencing at the beginning of streptozotocin-induced diabetes at the age of eight weeks. This treatment, previously demonstrated to increase free adenosine levels in vivo, was maintained until the age of 16weeks. Retinal inflammation was evaluated using Western blot, Real-Time PCR and immuno-staining analyses. Role of A2AAR signaling in the anti-inflammation effect of ABT-702 was analyzed in Amadori-glycated-albumin (AGA)-treated microglial cells. Key findingsAt 16weeks, when diabetic mice exhibit significant signs of retinal inflammation including up-regulation of oxidative/nitrosative stress, A2AAR, ENT1, Iba1, TNF-α, ICAM1, retinal cell death, and down-regulation of AK, the ABT-702 treated group showed lower signs of inflammation compared to control animals receiving the vehicle. The involvement of adenosine signaling in the anti-inflammation effect of ABT-702 was supported by the TNF-α release blocking effect of A2AAR antagonist in AGA-treated microglial cells. SignificanceThese results suggest a role for AK in regulating adenosine receptor signaling in the retina. Inhibition of AK potentially amplifies the therapeutic effects of site- and event-specific accumulation of extracellular adenosine, which is of highly translational impact.

Abstract 5412: Nuclear AMPKα2 is a repressor of alternative splicing of a subset of cancer-associated genes.

Abstract Alternative splicing of pre-mRNA is a fundamental cellular process that selectively joins alternate exons together to produce isoforms that differ in sequence, thereby significantly contributing to proteomic complexity as well as to post-transcriptional gene regulations. More than 90% of human genes, including oncogenes and tumor suppressor genes, have been predicted to undergo alternative splicing. Alternatively spliced isoforms function differently, sometimes antagonistically, and often exist in a delicate equilibrium that is perturbed during tumorigenesis. Previously, using caffeine as a tool, we have revealed a mechanism for alternative splicing regulation of a subset of cancer-associated genes, and have shown that this regulation is mediated, at least in part, by splicing factor SFRS2 (sc35) [Shi et al., MCB 28(2):883-895,2008]. In the present study, we found that the nucleoside adenosine, which is structurally similar to caffeine, can inhibit the caffeine-mediated and sc35-induced alternative splicing. This inhibition requires the function of adenosine transporters, suggesting an intracellular adenosine-mediated mechanism rather than adenosine receptor-mediated mechanisms. In addition, Iodotubercidin (Itu), an inhibitor of adenosine kinase, was found to inhibit the caffeine-mediated splicing as well. Since both caffeine and adenosine are known for roles in metabolic homeostasis, we specifically examined AMPKα, the catalytic subunit of the heterotrimeric AMPK (5’AMP-activated protein kinase) that is encoded by two genes: α1 and α2. Surprisingly, while RNAi-knockdown of AMPKα1 slightly reduced the basal-level as well as the caffeine-induced alternative splicing, AMPKα2-knockdown significantly increased alternative splicing of a reporter minigene. Importantly, neither caffeine nor adenosine significantly changed the level of AMPKα1 and α2. However, immunofluorescence studies revealed that AMPKα2 was predominantly localized in the nucleus in the presence of adenosine while largely cytoplasmic following caffeine treatment. Taken together, we propose that nuclear AMPKα2 plays a repressive role in the regulation of alternative splicing. Cancer is being increasingly recognized as a metabolic disease. AMPK is a “master switch” that senses cellular energy levels to rewire cellular metabolism accordingly. A great number of cancer-specific splice variants have been observed in both the laboratory and the clinic, and some of these splice variants are believed to be involved in the etiology and/or maintenance of the cancer phenotype. The identification of a role for AMPKα2 in alternative splicing regulation suggests another link between cellular metabolism and regulation of gene expression in cancer. Further studies will shed light on the largely unrevealed regulatory network linking alternative splicing, metabolism and cancer (This work is supported by NCI RO-1 CA122573) Citation Format: Jia Shi, Barton Kamen, Kathleen W. Scotto. Nuclear AMPKα2 is a repressor of alternative splicing of a subset of cancer-associated genes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5412. doi:10.1158/1538-7445.AM2013-5412

6-Alkyl-, 6-aryl- or 6-hetaryl-7-deazapurine ribonucleosides as inhibitors of human or MTB adenosine kinase and potential antimycobacterial agents

Title 6-alkyl-, 6-aryl- and 6-hetaryl-7-deazapurine ribonucleosides previously known as nanomolar cytostatics were found to be potent inhibitors of either human or mycobacterial (MTB) adenosine kinase (ADK). Several new derivatives bearing bulky substituents at position 6 were non-cytotoxic but selectively inhibited MTB ADK. However, most of the nucleosides (ADK inhibitors) as well as their octadecylphosphate prodrugs were inactive in the whole cell assay of inhibition of Mycobacterium bovis growth. 6-Methyl-7-deazapurine ribonucleoside was found to be a potent antimycobacterial agent.

Involvement of Akt2/protein kinase B β (PKBβ) in the 8‐Cl‐cAMP‐induced cancer cell growth inhibition

8-chloro-cyclic AMP (8-Cl-cAMP), which induces differentiation, growth inhibition, and apoptosis in various cancer cells, has been investigated as a putative anti-cancer drug. However, the exact mechanism of 8-Cl-cAMP functioning in cancer cells is not fully understood. Akt/protein kinase B (PKB) genes (Akt1, Akt2, and Akt3) encode enzymes belonging to the serine/threonine-specific protein kinase family. It has been suggested that Akt/PKB enhances cell survival by inhibiting apoptosis. Recently, we showed that 8-Cl-cAMP and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) inhibited cancer cell growth through the activation of AMPK and p38 MAPK. Therefore, we anticipated that the phosphorylation of Akt/PKB would be decreased upon treatment with 8-Cl-cAMP. However, treatment with 8-Cl-cAMP and AICAR induced the phosphorylation of Akt/PKB, which was inhibited by ABT702 (an adenosine kinase inhibitor) and NBTI (an adenosine transporter inhibitor). Furthermore, whereas Compound C (an AMPK inhibitor), AMPK-DN (AMPK-dominant negative) mutant, and SB203580 (a p38 MAPK inhibitor) did not block the 8-Cl-cAMP-induced phosphorylation of Akt/PKB, TCN (an Akt1/2/3 specific inhibitor) and an Akt2/PKBβ-targeted siRNA inhibited the 8-Cl-cAMP- and AICAR-mediated phosphorylation of AMPK and p38 MAPK. TCN also reversed the growth inhibition mediated by 8-Cl-cAMP and AICAR. Moreover, an Akt1/PKBα-targeted siRNA did not reduce the phosphorylation of AMPK and p38 MAPK after treatment with 8-Cl-cAMP. These results suggest that Akt2/PKBβ activation promotes the phosphorylation of AMPK and p38 MAPK during the 8-Cl-cAMP- and AICAR-induced growth inhibition.

Extracellular guanosine regulates extracellular adenosine levels

The aim of this investigation was to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels. Rat preglomerular vascular smooth muscle cells were incubated with adenosine, guanosine, or both. Guanosine (30 μmol/l) per se had little effect on extracellular adenosine levels. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) were 0.125 ± 0.020 μmol/l, indicating rapid disposition of extracellular adenosine. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) plus guanosine (30 μmol/l) were 1.173 ± 0.061 μmol/l, indicating slow disposition of extracellular adenosine. Cell injury increased extracellular levels of endogenous adenosine and guanosine, and the effects of cell injury on endogenous extracellular adenosine were modulated by altering the levels of endogenous extracellular guanosine with exogenous purine nucleoside phosphorylase (converts guanosine to guanine) or 8-aminoguanosine (inhibits purine nucleoside phosphorylase). Extracellular guanosine also slowed the disposition of extracellular adenosine in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts, and kidney epithelial cells and in human aortic and coronary artery vascular smooth muscle cells and coronary artery endothelial cells. The effects of guanosine on adenosine levels were not mimicked or attenuated by 5-iodotubericidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)-adenine (adenosine deaminase inhibitor), 5-aminoimidazole-4-carboxamide (guanine deaminase inhibitor), aristeromycin (S-adenosylhomocysteine hydrolase inhibitor), low sodium (inhibits concentrative nucleoside transporters), S-(4-nitrobenzyl)-6-thioinosine [inhibits equilibrative nucleoside transporter (ENT) type 1], zidovudine (inhibits ENT type 2), or acadesine (known modulator of adenosine levels). Guanosine also increases extracellular inosine, uridine, thymidine, and cytidine, yet decreases extracellular uric acid. In conclusion, extracellular guanosine regulates extracellular adenosine levels.

Pharmacochaperoning of the ER-retained A1adenosine receptor

Background The A1 adenosine receptor is a member of the rhodopsinrelated subfamily of GPCRs. Point mutations in the conserved NPxxY(x)5,6F motif at the junction of helix 7 and the C-terminus disrupt surface targeting of the receptor and result in its intracellular retention. This trafficking arrest can be overcome by addition of receptor ligands (pharmacochaperoning) that stabilize the receptor fold and thus promote surface expression. The mutants serve as a tool to explore a ramification of the retaliatory metabolite complex: hypoxia leads to intracellular accumulation of adenosine (by breakdown of ATP and by inhibition of adenosine kinase). Intracellular and extracellular adenosine levels are in equilibrium because of the action of the equilibrative nucleoside transporters. Extracellular adenosine dampens cellular metabolism by acting on inhibitory A1 adenosine receptors and thus counteracts the impact of hypoxia. If adenosine also pharmacochaperoned A1 adenosine receptors during hypoxia, it would enhance its effectiveness as a protective agent.

Stimulation of adenosine receptors in the nucleus accumbens reverses the expression of cocaine sensitization and cross-sensitization to dopamine D2 receptors in rats

Adenosine receptors co-localize with dopamine receptors on medium spiny nucleus accumbens (NAc) neurons where they antagonize dopamine receptor activity. It remains unclear whether adenosine receptor stimulation in the NAc restores cocaine-induced enhancements in dopamine receptor sensitivity. The goal of these studies was to determine whether stimulating A1 or A2A receptors in the NAc reduces the expression of cocaine sensitization. Rats were sensitized with 7 daily treatments of cocaine (15 mg/kg, i.p.). Following one-week withdrawal, the effects of intra-NAc microinjections of the adenosine kinase inhibitor (ABT-702), the adenosine deaminase inhibitor (deoxycoformycin; DCF), the specific A1 receptor agonist (CPA) and the specific A2A receptor agonist (CGS 21680) were tested on the behavioral expression of cocaine sensitization. The results indicate that intra-NAc pretreatment of ABT-702 and DCF dose-dependently blocked the expression of cocaine sensitization while having no effects on acute cocaine sensitivity, suggesting that upregulation of endogenous adenosine in the accumbens is sufficient to non-selectively stimulate adenosine receptors and reverse the expression of cocaine sensitization. Intra-NAc treatment of CPA significantly inhibited the expression of cocaine sensitization, which was reversed by both A1 and A2A receptor antagonism. Intra-NAc treatment of CGS 21680 also significantly inhibited the expression of cocaine sensitization, which was selectively reversed by A2A, but not A1, receptor antagonism. Finally, CGS 21680 also inhibited the expression of quinpirole cross-sensitization. Together, these findings suggest that adenosine receptor stimulation in the NAc is sufficient to reverse the behavioral expression of cocaine sensitization and that A2A receptors blunt cocaine-induced sensitization of postsynaptic D2 receptors.

Adenosine augmentation ameliorates psychotic and cognitive endophenotypes of schizophrenia

An emerging theory of schizophrenia postulates that hypofunction of adenosine signaling may contribute to its pathophysiology. This study was designed to test the "adenosine hypothesis" of schizophrenia and to evaluate focal adenosine-based strategies for therapy. We found that augmentation of adenosine by pharmacologic inhibition of adenosine kinase (ADK), the key enzyme of adenosine clearance, exerted antipsychotic-like activity in mice. Further, overexpression of ADK in transgenic mice was associated with attentional impairments linked to schizophrenia. We observed that the striatal adenosine A2A receptor links adenosine tone and psychomotor response to amphetamine, an indicator of dopaminergic signaling. Finally, intrastriatal implants of engineered adenosine-releasing cells restored the locomotor response to amphetamine in mice overexpressing ADK, whereas the same grafts placed proximal to the hippocampus of transgenic mice reversed their working memory deficit. This functional double dissociation between striatal and hippocampal adenosine demonstrated in Adk transgenic mice highlights the independent contributions of these two interconnected brain regions in the pathophysiology of schizophrenia and thus provides the rationale for developing local adenosine augmentation therapies for the treatment of schizophrenia.

Activation of AMP-Activated Protein Kinase Inhibits the Proliferation of Human Endothelial Cells

AMP-activated protein kinase (AMPK) is an evolutionary conserved energy-sensing enzyme that regulates cell metabolism. Emerging evidence indicates that AMPK also plays an important role in modulating endothelial cell function. In the present study, we investigated whether AMPK modulates endothelial cell growth. Treatment of cultured human umbilical vein endothelial cells with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 6,7-dihydro-4-hydroxy-3-(2'-hydroxy[1,1'-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662), or metformin inhibited cell proliferation and DNA synthesis. The antiproliferative action of AICAR was largely prevented by the adenosine kinase inhibitor 5'-iodotubercidin and mimicked by infecting endothelial cells with an adenovirus expressing constitutively active AMPK. In contrast, pharmacological blockade of endothelial nitric oxide synthase or heme oxygenase-1 activity failed to reverse the inhibition of endothelial cell growth by AICAR. Flow cytometry experiments revealed that pharmacological activation of AMPK arrested endothelial cells in the G₀/G₁ phase of the cell cycle, and this was associated with increases in p53 phosphorylation and p53, p21, and p27 protein expression and decreases in cyclin A protein expression and retinoblastoma protein phosphorylation. In addition, silencing p21 and p27 expression partially restored the mitogenic response of AMPK-activated cells. Finally, activation of AMPK by AICAR blocked the migration of endothelial cells after scrape injury and stimulated tube formation by endothelial cells plated onto Matrigel-coated plates. In conclusion, these studies demonstrate that AMPK activation inhibits endothelial cell proliferation by elevating p21 and p27 expression. In addition, they show that AMPK regulates endothelial cell migration and differentiation and identify AMPK as an attractive therapeutic target in treating diseases associated with aberrant endothelial cell growth.