BACKGROUND: In two-kidney one clip Goldbaltt hypertensive rats(2K1C GHR), clipped kidney may be exposed to low pressure and unclipped kidney to high pressure. In addition, both kidneys may have a different amount of adenosine which is increased by ischemia and plays an important role for renin release. The aim of this study was to invstigate the responsmiveness for renin release to adenosine agonists and antagonist in clipped and unclipped kidney of 2K1C GHR. METHODS: Emplying kidney slices from both unclipped and unclipped kidney of 2K1C GHR, the alteration by adenosine agonists and antagonist of renin release was studied. RESULTS: The renal renin content and basal renin release from unclipped kidney slices were suppressed, whereas those from clipped kidney were augmented Adenosine Al receptor agonist, cyclohexyladenosne(CHA), phenylisopropyl adenosine(PIA) and adenosine caused a decrease in renin release from clipped kidney slices. Adenosine A2 receptor agonist, NECA, and nonspecific adenosine receptor aganist, 2-chloroadenosine(CA) caused an increase in renin release from clipped kidney slices. Adenosine receptor antagonist, 8-phenyltheophylline(8-PT) caused an increase in renin release from clipped kidney slices. In unclipped kidney, however, the renin release in response to NECA, CA or 8-PT was reversed and the decreasing effect of renin release to CHA and adenosine was slightly inereased. CONCLUSION: These results suggest that the responsiveness of adenosine receptors, which may participate in renin release is modified in clipped and unclipped kidney of 2K1C GHR.
Adenosine* ; Adenosine-5'-(N-ethylcarboxamide) ; Animals ; Hypertension, Renovascular ; Ischemia ; Kidney ; Rats* ; Receptors, Adenosine A2 ; Receptors, Purinergic P1 ; Renin*

BACKGROUND: Intercellular adhesion molecule(ICAM)-1 mediates cell to cell adhesion by acting as a receptor for leukocyte surface antigen. Increased ICAM-1 expression was observed in chronic inflammatory skin diseases, such as psoriasis, atopic dermatitis and allergic contact dermatitis. Adenosine is an endogenous antiinflammatory agent released by cells under metabolically unfavorable conditions, recently the studies about antiinflammatory effects of adenosine in various tissues were increased, but there are few studies about the effect of adenosine on epidermal keratinocytes. OBJECTIVE: We investigated the effects of adenosine on ICAM-1 expression in cultured human keratinocyte cell line HaCaT cells. METHODS: Our study analyses the ICAM-1 expression in HaCaT cells by various stimulants and the effects of adenosine, adenosine receptor agonist&antagonist and an inhibitor of cellular adenosine uptake on ICAM-1 expression of cells stimulated by IFN-gamma through the cell-ELISA (enzyme-linked immunosorbent assay)&FACS (fluorescence-activated cell sorter) analysis. RESULTS: The results are summerized as follows: 1. ICAM-1 expression was significantly increased by IFN-gamma(500U/ml), IFN-gamma&TNF-alpha(10-8M) and IFN-gamma&LPS(10-8M)(p<0.05), but not by TNF-alpha, LPS and TNF-alpha & LPS. 2. Incubation of HaCaT cells with IFN-gamma(1-2000U/ml) for 48 hours induced dose-dependent expression of ICAM-1 at above 500U/ml of IFN-gamma. 3. Adenosine had no effect on ICAM-1 expression of unstimulated cells. 4. Adenosine(esp. 10-4M) significantly inhibited ICAM-1 expression of cells stimulated by IFN-gamma(p<0.01). 5. Adenosine(10-4M) significantly inhibited ICAM-1 expression of cells stimulated by IFN-gamma(p<0.01), IFN-gamma & TNF-alpha(p<0.05) and IFN-gamma&LPS. 6. The inhibition of ICAM-1 expression was not observed when cells were preincubated with an adenosine A1 receptor agonist(R-PIA) or an adenosine A2 receptor agonist (NECA). 7. The inhibition of ICAM-1 expression of adenosine was not affected by pretreatment state with an adenosine A1 & A2 receptor antagonist(theophylline)(p<0.01). 8. The inhibition of ICAM-1 expression of adenosine was not observed by pretreatment state with an inhibitor of adenosine cellular uptake (dipyridamole). CONCLUSION: High concentration of adenosine inhibits enhanced ICAM-1 expression on HaCaT cells by stimulated with IFN-gamma and these inhibitory effects of adenosine are mediated through other adenosine receptors excect adenosine A1 and A2 receptors. And we suggest that there may be an unknown intracellular mechanism about inhibition of ICAM-1 expression via intracellular-uptaken adenosine.
Adenosine* ; Antigens, Surface ; Cell Adhesion ; Cell Line* ; Dermatitis, Allergic Contact ; Dermatitis, Atopic ; Humans* ; Intercellular Adhesion Molecule-1* ; Keratinocytes* ; Leukocytes ; Psoriasis ; Receptor, Adenosine A1 ; Receptors, Adenosine A2 ; Receptors, Purinergic P1 ; Skin Diseases ; Tumor Necrosis Factor-alpha

To establish a model of inactivation adenosine A2A receptors in brain tissues of mice, we transplanted bone marrow cells (BMCs) from wild type (WT) C57BL/6 mice into A2A receptor knockout (A2A KO) C57BL/6 mice which were previously fractionated total body irradiation of 6.2 Gyx2. Six weeks later, we identified and evaluated the model. The results showed that the sexual chromagene pattern on white blood cells of recipient mice changed from female pattern to male pattern and there were 95.9% of A2AR+ cells in peripheral white blood cells of recipient mice, whereas there was no significant difference of A2AR mRNA level in brains between these recipient mice and A2AR KO mice. Furthermore, there was no significant difference of breathing frequency, brain water content and level of glutamate between the model mice and WT mice. These results indicated that we established successfully a mouse model of inactivation adenosine A2A receptors in brain tissues. This may provide a new and efficient strategy to study the effect of adenosine A2A receptors in disease and injuries of central nervous system.
Animals ; Bone Marrow Transplantation ; Brain ; metabolism ; Disease Models, Animal ; Female ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Receptors, Adenosine A2 ; deficiency ; genetics ; Whole-Body Irradiation

Polydeoxyribonucleotide (PDRN) is safe and effective in wound healing, cellular growth, synthesis of extracellular matrix protein, and inflammation reduction via activation of adenosine A2 receptors. We report a 28-year-old male patient treated with PDRN injections for chronic non-healing wound refractory to negative pressure wound therapy, skin graft, or growth factors. Three injections of PDRN were administered at the wound site into the anterior and medial sides of the left stump on the 1st, 4th, and 9th days of hospitalization. The PDRN ameliorated wound healing by enhancing cell growth, tissue repair, and angiogenesis. PDRN application represents a potential treatment for non-healing wounds obviating the need for additional therapies, and hospitalization, as well as improve patient’s activities of daily living.
Activities of Daily Living ; Adult ; Amputees* ; Extracellular Matrix ; Hospitalization ; Humans ; Inflammation ; Intercellular Signaling Peptides and Proteins ; Male ; Negative-Pressure Wound Therapy ; Polydeoxyribonucleotides ; Receptors, Adenosine A2 ; Skin ; Transplants ; Wound Healing ; Wounds and Injuries*

PURPOSE: Adenosine triphosphate (ATP) evokes several cellular responses in microglia including propagation. However, the role of the purinoceptor on ROS generation in microglia is unclear. In order to determine the action of the purinoceptor in microglia, the effects of ATP on ROS generation and cellular proliferation in BV-2 murine microglial cells were evaluated. An additional aim of this study was to investigate signal transduction pathways using several inhibitors. METHODS: The [Ca2+] was measured using Ca2+ sensitive indicator, Fura-2/AM. ROS production was observed by fluorescence-confocal microscope and cell proliferation was evaluated by counting cell number. RESULTS: ATP increased the intracellular calcium levels ([Ca2+]i) in BV-2 cells in a dose-dependent manner. This increase was attenuated by pretreatment with a calcium chelator (EGTA) and a phospholipase C (PLC) inhibitor (U-73122) while the protein tyrosine kinase (PTK) inhibitor (genistein) had no inhibitory effects. To identify the effects of the nucleotides, ROS generation was observed in the nucleotide-stimulated BV-2 cells. The treatment with 100 M ATP induced ROS generation, but 100 M adenosine and 100 M UTP did not. To investigate the signal transduction pathway in ATP-induced ROS generation, several inhibitors were pretreated before adding ATP. ATP- induced ROS production was blocked by pretreatment with either 0.5 mM EGTA or 10 M U73122 while 40 M genistein had an inhibitory effect on ATP action. Correspondingly, 40 M KN62 (CaM kinase II inhibitor), 1 M sphingosine (protein kinase C inhibitor), 1 nM DPI (NADPH oxidase inhibitor) and 50 M mepacrine (phospholipase A2 inhibitor) could suppress ATP-induced ROS generation. The effects of ATP on cell proliferation was observed 3 days after ATP treatment and its peak velocity after 4 days. NF-kB activation was observed after the cells were incubated with 0.1 mM ATP. The maximal level of NF-kB activation was obtained with 0.3 mM ATP while higher concentrations were less effective. CONCLUSION: Overall, we conclude that ATP in BV-2 cells induces ROS generation and cell propagation. The signal transduction pathways including calcium, CaM kinase II, PLC, protein kinase C, phospholipase A2 and NADPH oxidase are involved in ATP-induced ROS generation.
Adenosine ; Adenosine Triphosphate* ; Calcium ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Cell Count ; Cell Proliferation* ; Egtazic Acid ; Genistein ; Microglia ; NADPH Oxidase ; NF-kappa B ; Nucleotides ; Oxidoreductases ; Phospholipases A2 ; Phosphotransferases ; Protein Kinase C ; Protein-Tyrosine Kinases ; Quinacrine ; Reactive Oxygen Species* ; Receptors, Purinergic ; Signal Transduction ; Sphingosine ; Type C Phospholipases ; Uridine Triphosphate

Platelet aggregation is not only an essential part of hemostasis, but also initiates acute coronary syndrome or ischemic stroke. The precise understanding of the activation mechanism of platelet aggregation is fundamental for the development of more effective agents against platelet aggregation. Adenosine diphosphate, thrombin, and thromboxane A2 activate platelet integrin alphaIIbbeta3 through G protein-coupled receptors. G protein-mediated signaling pathways, which are initiated by Gq, G12/G13 or Gi, include phospholipase C with calcium signaling, Rho signaling, protein kinase C and phosphatidylinositol 3-kinase. Rap1b, Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor I, Rap1-GTP-interacting adaptor molecule, and Akt are important proteins involved in G protein-mediated activation of integrin alphaIIbbeta3. Binding of talin-1 and kindlin-3 to cytoplasmic domains of beta3-integrin triggers a conformational change in the extracellular domains that increases its affinity for ligands, such as fibrinogen or von Willebrand factor. Fibrinogens act as bridges between adjacent platelets to generate a platelet aggregate.
Acute Coronary Syndrome ; Adenosine Diphosphate ; Blood Platelets ; Calcium Signaling ; Cytoplasm ; Fibrinogen ; Guanine Nucleotide Exchange Factors ; Hemostasis ; Ligands ; Phosphatidylinositol 3-Kinase ; Platelet Activation ; Platelet Aggregation ; Platelet Glycoprotein GPIIb-IIIa Complex ; Protein Kinase C ; Proteins ; Receptors, G-Protein-Coupled ; Stroke ; Thrombin ; Thromboxane A2 ; Type C Phospholipases ; von Willebrand Factor

OBJECTIVE: To explore the role of the low-affinity A2b adenosine receptors (Adora2b) in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide and its mechanism. METHODS: Rat pulmonary microvascular endothelial cells (PMVECs) were isolated and cultured in vitro. After serum deprivation for 24 hours, cells were pretreated with Adora2b specific agonist BAY60-6583 (0.1, 1, 10 μmol/L) or Adora2b specific antagonist PSB1115 (1 μmol/L) for 1 hour, respectively, and then challenged with LPS (100 μg/L). Cells without treatment were served as the control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. After incubation with specific drugs for 24 hours, the apoptosis of PMVECs was analyzed by flow cytometry using Annexin V/propidium iodide (PI) technique. The levels of early inflammatory factors in cultured medium were measured using enzyme linked immunosorbent assay (ELISA). The mRNA expressions of chemotactic factors and adhesion molecules were determined by real-time quantitative-polymerase chain reaction (RT-qPCR). Polymorph nuclear neutrophils (PMNs) from venous blood of healthy rats were isolated, and PMN migration through PMVECs monolayer under stimulation of drugs was observed in transwell inserts. The monolayer permeability of PMVECs after adhesion of PMNs was determined by fluorescein isothiocyanate (FITC)-albumin assay. Oxidative stress was detected by DCFH-DA assay. RESULTS: Compared with the control group, more cells entered into the apoptosis stage after LPS challenge. Meanwhile, the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in cultured medium were significantly increased, as well as the mRNA expressions of chemotactic factors [C-X-C motif chemokine ligand 1 (CXCL-1), CXCL-3 and monocyte chemoattractant protein-1 (MCP-1)] and adhesion molecules [E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. More PMNs migrated through PMVECs following adhesion and the monolayer permeability of PMVECs was rapidly enhanced. The oxidative stress was upregulated. Compared with LPS group, BAY60-6583 pretreatment could dose-dependently decrease the rate of apoptosis, attenuate trans-endothelial migration of PMNs and decrease the endothelial cell barrier leakage. There were significant differences even after incubation of 0.1 μmol/L BAY60-6583 [apoptosis rate: (21.12±2.12)% vs. (27.66±3.57)%, number of migrated PMNs/HP: 260.60±18.24 vs. 290.20±16.48, permeability coefficient (Pd, ×10^-6 cm/s): 28.28±2.04 vs. 32.55±2.13, all P < 0.05]. Meanwhile, BAY60-6583 pretreatment also downregulated the levels of early proinflammatory factors in a dose-dependent manner as well as the mRNA expressions of chemotactic factors and adhesion molecules. The statistic difference was significant while treated with 1 μmol/L BAY60-6583 [IL-1β (ng/L): 475.75±63.15 vs. 755.25±67.42, TNF-α (ng/L): 560.25±69.96 vs. 818.75±60.92, CXCL-1 mRNA (2^-ΔΔCt): 3.57±0.28 vs. 5.27±0.69, CXCL-3 mRNA (2^-ΔΔCt): 4.56±0.48 vs. 7.32±0.54, MCP-1 mRNA (2^-ΔΔCt): 2.21±0.31 vs. 3.35±0.21, E-selectin mRNA (2^-ΔΔCt): 4.64±0.09 vs. 7.28±0.73, ICAM-1 mRNA (2^-ΔΔCt): 4.14±0.30 vs. 5.89±0.25, VCAM-1 mRNA (2^-ΔΔCt): 2.23±0.19 vs. 2.92±0.33, all P < 0.05]. Furthermore, pretreatment of 10 μmol/L BAY60-6583 could decrease the oxidative stress [reactive oxygen species (RFU): 629.05±33.10 vs. 781.45±64.59, P < 0.05]. Contrast, PSB1115 pretreatment aggravated apoptosis of PMVECs after LPS incubation [(34.36±4.57)% vs. (27.66±3.57)%], upregulated expressions of proinflammatory and chemotactic factors as well as adhesion molecules [IL-1β (ng/L): 889.00±63.11 vs. 755.25±67.42, TNF-α (ng/L): 939.00±43.44 vs. 818.75±60.92, CXCL-1 mRNA (2^-ΔΔCt): 6.66±0.65 vs. 5.27±0.69, CXCL-3 mRNA (2^-ΔΔCt): 10.42±0.51 vs. 7.32±0.54, MCP-1 mRNA (2^-ΔΔCt): 4.85±0.34 vs. 3.35±0.21, E-selectin mRNA (2^-ΔΔCt): 8.42±0.47 vs. 7.28±0.73, ICAM-1 mRNA (2^-ΔΔCt): 7.46±0.72 vs. 5.89±0.25, VCAM-1 mRNA (2^-ΔΔCt): 4.35±0.26 vs. 2.92±0.33], aggravated trans-endothelial migration of PMNs (cells/HP: 348.40±22.68 vs. 290.20±16.48), enhanced the leakage of PMVECs monolayer [Pd (×10^-6 cm/s): 39.65±2.69 vs. 32.55±2.13] and increased oxidative stress in PMVECs [reactive oxygen species (RFU): 847.04±29.26 vs. 781.45±64.59], with statistically significant difference (all P < 0.05). CONCLUSIONS Activation of endothelial Adora2b attenuates LPS-induced pulmonary microvascular inflammation by decreasing the release of early inflammatory factors, downregulating expressions of chemotactic factors and adhesion molecules, attenuating trans-endothelial migration of PMNs and oxidative stress in PMVECs, which suggest endothelial Adora2b is apotential anti-inflammatory target in the treatment of LPS-induced acute lung injury.
Animals ; Endothelial Cells ; Inflammation ; Lipopolysaccharides/metabolism* ; Pneumonia ; Rats ; Receptor, Adenosine A2B/metabolism* ; Tumor Necrosis Factor-alpha

Numerous studies have shown that adenosine or adenosine agonists can stimulate angiogenesis. However, the effect of caffeine (a known adenosine receptor antagonist) on angiogenesis has not been previously studied. Accordingly, this study was undertaken to examine the effect of caffeine on angiogenesis and to clarify the mechanism involved. Chick chorioallantoic membrane assays were used to investigate the effect of caffeine on angiogenesis and proliferation assays using human umbilical vein endothelial cells (HUVECs), were used to study its effects on specific aspects of angiogenesis. The expressions of caspase-3 and Bcl-2 were examined by western blotting, immunofluorescence staining was used to identify HUVEC morphological changes, and fluorescence activated cell sorting (FACS) and DAPI staining were used to detect HUVEC apoptosis. Caffeine was found to inhibit blood vessel formation dose-dependently and to inhibit the proliferation of HUVECs time- and dose-dependently. FACS analysis and DAPI staining showed that inhibitory effect of caffeine on HUVEC proliferation was the result of apoptosis and the up-regulation of thrombospondin-1 (TSP-1). Furthermore, TSP-1 levels were down-regulated by NECA but were unaffected by CGS21680, indicating that caffeine regulated TSP-1 expression via adenosine A2B receptor. In addition, caffeine up-regulated caspase-3 and down-regulated Bcl-2 at the protein level. These results suggest that the inhibitory effect of caffeine on angiogenesis is associated, at least in part, with its induction of endothelial cell apoptosis, probably mediated by a caspase-3 dependent mechanism.
Adenosine ; Adenosine-5'-(N-ethylcarboxamide) ; Apoptosis ; Blood Vessels ; Blotting, Western ; Caffeine ; Caspase 3 ; Chorioallantoic Membrane ; Endothelial Cells ; Flow Cytometry ; Fluorescent Antibody Technique ; Glycosaminoglycans ; Human Umbilical Vein Endothelial Cells ; Indoles ; Phenethylamines ; Receptor, Adenosine A2B ; Receptors, Purinergic P1 ; Thrombospondin 1 ; Up-Regulation

Adenosine is a metabolite produced abundantly in the tumor microenvironment, dampening immune response in inflamed tissues via adenosine A2A receptor (A2AR) which is widely expressed on immune cells, inhibiting anti-tumor immune response accordingly. Therefore, blocking adenosine signaling pathway is of potential to promote anti-tumor immunity. This review briefly introduces adenosine signaling pathway, describes its role in regulating tumor immunity and highlights A2AR blockade in cancer therapy. Prospective anti-tumor activity of adenosine/A2AR inhibition has been revealed by preclinical data, and a number of clinical trials of A2AR antagonists are under way. Primary results from clinical trials suggest that A2AR antagonists are well tolerated in cancer patients and are effective both as monotherapy and in combination with other therapies. In the future, finding predictive biomarkers are critical to identify patients most likely to benefit from adenosine pathway blockade, and further researches are needed to rationally combine A2AR antagonists with other anti-tumor therapies.
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Adenosine/therapeutic use* ; Adenosine A2 Receptor Antagonists/therapeutic use* ; Humans ; Lung Neoplasms ; Receptor, Adenosine A2A/metabolism* ; Tumor Microenvironment

OBJECTIVETo establish a mice model with selective inactivation adenosine A2A receptors (A2ARs) in peripheral white blood cells (PWBC).

METHODSA2ARs were selectively inactivated in PWBCs by transplanting bone marrow cells (BMCs) from A2AR knockout (KO) mice into their wild type (WT) littermates after a single total body irradiation of 9.5 Gy or fractionated total body irradiation of 6.2 Gy x 2. The efficiency of reconstitution of bone marrow-derived cells in chimeric mice was assessed.

RESULTSPCR band patterns changed from the recipient pattern (one band of 330 bp) to the donor (two bands of 300 and 330 bp) pattern. Immunohistochemistry analysis showed that 10.21% of cells were A2AR+ in PWBCs in KO--> WT mice, whereas 96.72% of cells were A2AR+ in WT mice. The survival rates of mice irradiated with 6.2 Gy x 2 and transplanted with more than 6 x 10(6) BMCs were about 91%.

CONCLUSIONA murine model of selective inactivation adenosine A2A receptors in PWBCs was established successfully.