腺苷脱氨酶抑制巨噬细胞增殖迁移的作用研究

目的:探讨腺苷脱氨酶对鼠源巨噬细胞RAW264.7增殖、迁移、细胞周期、细胞凋亡的影响。方法:用不同浓度(0、0.25、1.25、2.5、5U/m L)的腺苷脱氨酶处理RAW264.7细胞后,用实时细胞分析系统检测细胞增殖能力,用流式细胞术检测腺苷脱氨酶对细胞凋亡和周期的影响,划痕修复实验检测RAW264.7细胞迁移能力。结果:与对照组相比,高浓度腺苷脱氨酶(2.5 U/m L、5 U/m L)处理可以显著抑制RAW264.7细胞的增殖能力,且抑制效果随腺苷脱氨酶浓度升高而增强(P<0.05)。流式细胞术检测结果显示,相较于对照组,高浓度腺苷脱氨酶(2.5 U/m L)处理可以诱导RAW264.7细胞凋亡,并导致细胞周期G2/M期阻滞(P<0.05)。此外,细胞划痕实验表明,高浓度腺苷脱氨酶(2.5 U/m L)处理可以显著抑制RAW264.7巨噬细胞的迁移能力(P<0.05)。结论:高浓度腺苷脱氨酶对巨噬细胞增殖和迁移具有抑制作用,并可诱导细胞凋亡和细胞周期阻滞。     

Molecular probes for the human adenosine receptors

Adenosine receptors, G protein–coupled receptors (GPCRs) that are activated by the endogenous ligand adenosine, have been considered potential therapeutic targets in several disorders. To date however, only very few adenosine receptor modulators have made it to the market. Increased understanding of these receptors is required to improve the success rate of adenosine receptor drug discovery. To improve our understanding of receptor structure and function, over the past decades, a diverse array of molecular probes has been developed and applied. These probes, including radioactive or fluorescent moieties, have proven invaluable in GPCR research in general. Specifically for adenosine receptors, the development and application of covalent or reversible probes, whether radiolabeled or fluorescent, have been instrumental in the discovery of new chemical entities, the characterization and interrogation of adenosine receptor subtypes, and the study of adenosine receptor behavior in physiological and pathophysiological conditions. This review summarizes these applications, and also serves as an invitation to walk another mile to further improve probe characteristics and develop additional tags that allow the investigation of adenosine receptors and other GPCRs in even finer detail.

     

Adenosine A1 and A2A receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices

6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson’s disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 μM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A₁ (A₁R) and/or A_2A receptors (A_2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A₁R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A₁R agonist, did not alter GUO effects. Regarding A_2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A_2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A₁R antagonist DPCPX, and the A_2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A₁R-A_2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

     

Purine Modulation of Cytokine Release During Diuretic Therapy of Rheumatoid Arthritis

Since free radicals are implicated in rheumatoid arthritis (RA) and since uric acid is a free radical scavenger, we examined the effects of treating RA patients with with the diuretic bumetanide to try to improve their arthritic control. Seventy patients, aged 18–75 years, were randomised to receive bumetanide 4 mg/day or placebo. Uric acid levels increased, but not that of other purines, in the blood of drug‐treated patients compared with placebo‐treated controls. There were no significant changes in clinical measurements of disease activity or in ESR or CRP levels. There were no over all differences in the blood levels of the cytokines, nor in the basal or stimulated production of cytokines from the blood cultures. The adenosine receptor agonist 5′Nethylcarboxamido‐ adenosine (NECA) used to modify cytokine release in cultures of whole blood taken from the patients, depressed the release of tumour necrosis factor‐a (TNFa), but failed to depress the release of interleukin‐1b (IL‐1b) or interleukin‐6 (IL‐6), a difference from earlier studies of healthy control subjects and, thus, a difference which may contribute to the disease activity.     

Capsaicin as an inducer of damage-associated molecular patterns (DAMPs) of immunogenic cell death (ICD) in human bladder cancer cells

Few conventional cytotoxic anticancer therapeutics induce immunogenic cell death (ICD). This means that they induce tumor cells to undergo apoptosis while eliciting the emission of a spatiotemporal-defined combination of damage-associated molecular patterns (DAMPs) decoded by the immune system to activate antitumor immunity effective for long-term therapeutic success. The neurotoxin capsaicin (CPS) can induce both cancer cell apoptosis and immune-mediated tumor regression. In the present study, we investigated whether CPS is capable of eliciting the emission of ICD hallmarks in human bladder cancer cell lines undergoing apoptosis. We demonstrated that CPS induces pre- and early apoptotic cell surface exposure of calreticulin (CRT), HSP90, and HSP70 as well as ATP release. Moreover, CRT exposure was prevented by inhibition of endoplasmic reticulum–Golgi traffic by brefeldin A. Furthermore, high-mobility group box 1, HSP90, and HSP70 were passively released at late apoptotic stages. We provide the first evidence that CPS is an inducer of ICD hallmarks, suggesting CPS as a novel potential immunogenic cytotoxic agent.     

A study of the possible association between adenosine A2A receptor gene polymorphisms and attention‐deficit hyperactivity disorder traits

The adenosine A_2A receptor (ADORA2A) is linked to the dopamine neurotransmitter system and is also implicated in the regulation of alertness, suggesting a potential association with attention‐deficit hyperactivity disorder (ADHD) traits. Furthermore, animal studies suggest that the ADORA2A may influence ADHD‐like behavior. For that reason, the ADORA2A gene emerges as a promising candidate for studying the etiology of ADHD traits. The aim of this study was to examine the relationship between ADORA2A gene polymorphisms and ADHD traits in a large population‐based sample. This study was based on the Child and Adolescent Twin Study in Sweden (CATSS), and included 1747 twins. Attention‐deficit hyperactivity disorder traits were assessed through parental reports, and samples of DNA were collected. Associations between six single nucleotide polymorphisms (SNPs) and ADHD traits were examined, and results suggested a nominal association between ADHD traits and three of these SNPs: rs3761422, rs5751876 and rs35320474. For one of the SNPs, rs35320474, results remained significant after correction for multiple comparisons. These results indicate the possibility that the ADORA2A gene may be involved in ADHD traits. However, more studies replicating the present results are warranted before this association can be confirmed .     

Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the “adenosine hypothesis”

Schizophrenia (SZ) is a major chronic neuropsychiatric disorder characterized by a hyperdopaminergic state. The hypoadenosinergic hypothesis proposes that reduced extracellular adenosine levels contribute to dopamine D₂ receptor hyperactivity. ATP, through the action of ecto-nucleotidases, constitutes a main source of extracellular adenosine. In the present study, we examined the activity of ecto-nucleotidases (NTPDases, ecto-5′-nucleotidase, and alkaline phosphatase) in the postmortem putamen of SZ patients (n = 13) compared with aged-matched controls (n = 10). We firstly demonstrated, by means of artificial postmortem delay experiments, that ecto-nucleotidase activity in human brains was stable up to 24 h, indicating the reliability of this tissue for these enzyme determinations. Remarkably, NTPDase-attributable activity (both ATPase and ADPase) was found to be reduced in SZ patients, while ecto-5′-nucleotidase and alkaline phosphatase activity remained unchanged. In the present study, we also describe the localization of these ecto-enzymes in human putamen control samples, showing differential expression in blood vessels, neurons, and glial cells. In conclusion, reduced striatal NTPDase activity may contribute to the pathophysiology of SZ, and it represents a potential mechanism of adenosine signalling impairment in this illness.     

Adenosine A2A receptor (A2AR) is a fine-tune regulator of the collagen1:collagen3 balance

Adenosine is a potent endogenous anti-inflammatory and immunosuppressive metabolite that is a potent modulator of tissue repair. However, the adenosine A_2A receptor (A_2AR)-mediated promotion of collagen synthesis is detrimental in settings such as scarring and scleroderma. The signaling cascade from A_2AR stimulation to increased collagen production is complex and obscure, not least because cAMP and its downstream molecules PKA and Epac1 have been reported to inhibit collagen production. We therefore examined A_2AR-stimulated signaling for collagen production by normal human dermal fibroblasts (NHDF). Collagen1 (Col1) and collagen3 (Col3) content after A_2AR activation by CGS21680 was studied by western blotting. Contribution of PKA and Epac was analyzed by the PKA inhibitor PKI and by knockdowns of the PKA-Cα, -Cβ, -Cγ, Epac1, and Epac2. CGS21680 stimulates Col1 expression at significantly lower concentrations than those required to stimulate Col3 expression. A_2AR stimulates Col1 expression by a PKA-dependent mechanism since PKA inhibition or PKA-Cα and -Cβ knockdown prevents A_2AR-mediated Col1 increase. In contrast, A_2AR represses Col3 via PKA but stimulates both Col1 and Col3 via an Epac2-dependent mechanism. A_2AR stimulation with CGS21680 at 0.1 μM increased Col3 expression only upon PKA blockade. A_2AR activation downstream signaling for Col1 and Col3 expression proceeds via two distinct pathways with varying sensitivity to cAMP activation; more highly cAMP-sensitive PKA activation stimulates Col1 expression, and less cAMP-sensitive Epac activation promotes both Col1 and Col3 expression. These observations may explain the dramatic change in Col1:Col3 ratio in hypertrophic and immature scars, where adenosine is present in higher concentrations than in normal skin.     

Dehydration mediated microRNA response in the African clawed frog Xenopus laevis

Exposure to various environmental stresses induces metabolic rate depression in many animal species, an adaptation that conserves energy until the environment is again conducive to normal life. The African clawed frog, Xenopus laevis, is periodically subjected to arid summers in South Africa, and utilizes entry into the hypometabolic state of estivation as a mechanism of long term survival. During estivation, frogs must typically deal with substantial dehydration as their ponds dry out and X. laevis can endure > 30% loss of its body water. We hypothesize that microRNAs play a vital role in establishing a reversible hypometabolic state and responding to dehydration stress that is associated with amphibian estivation. The present study analyzes the effects of whole body dehydration on microRNA expression in three tissues of X. laevis. Compared to controls, levels of miR-1, miR-125b, and miR-16-1 decreased to 37 ± 6, 64 ± 8, and 80 ± 4% of control levels during dehydration in liver. By contrast, miR-210, miR-34a and miR-21 were significantly elevated by 3.05 ± 0.45, 2.11 ± 0.08, and 1.36 ± 0.05-fold, respectively, in the liver. In kidney tissue, miR-29b, miR-21, and miR-203 were elevated by 1.40 ± 0.09, 1.31 ± 0.05, and 2.17 ± 0.31-fold, respectively, in response to dehydration whereas miR-203 and miR-34a were elevated in ventral skin by 1.35 ± 0.05 and 1.74 ± 0.12-fold, respectively. Bioinformatic analysis of the differentially expressed microRNAs suggests that these are mainly involved in two processes: (1) expression of solute carrier proteins, and (2) regulation of mitogen-activated protein kinase signaling. This study is the first report that shows a tissue specific mode of microRNA expression during amphibian dehydration, providing evidence for microRNAs as crucial regulators of metabolic depression.