Expression Of Adenosine Receptor Subtypes Research Articles

Adenosine Receptors Expression in Human Retina and Choroid with Age-related Macular Degeneration.

Adenosine signaling modulates ocular inflammatory processes, and its antagonism mitigates neovascularization in both newborns and preclinical models of ocular neovascularization including age-related macular degeneration (AMD). The adenosine receptor expression patterns have not been well characterized in the human retina and choroid. Here we examined the expression of adenosine receptor subtypes within the retina and choroid of human donor eyes with and without AMD. Antibodies specifically targeting adenosine receptor subtypes A1, A2A, A2B, and A3 were used to assess their expression patterns. Quantitative real-time PCR analysis was used to confirm gene expression of these receptors within the normal human retina and choroid. We found that all four receptor subtypes were expressed in several layers of the retina, and within the retinal pigment epithelium and choroid. The expression of A1 receptors was more prominent in the inner and outer plexiform layers, where microglia normally reside, and supported by RNA expression in the retina. A2A and A2B showed similar expression patterns with prominent expression in the vasculature and retinal pigment epithelium. No dramatic differences in expression of these receptors were observed in eyes from patients with dry or wet AMD compared to control, with the exception A3 receptors. Eyes with dry AMD lost expression of A3 in the photoreceptor outer segments compared with eyes from control or wet AMD. The ocular presence of adenosine receptors is consistent with their proposed role in modulation of inflammation in both the retina and choroid, and their potential targeting for AMD treatment.

Estrogen stimulates adenosine receptor expression subtypes in human breast cancer MCF-7 cell line.

Estrogen is a steroid hormone that plays a key role in the development and regulation of reproductive system. It has been shown that estrogen is related to breast cancer development through binding to its receptors. In order to uncover the estrogen effects on adenosine receptor expression, estrogen-positive MCF-7 cells were used to treat with agonist and antagonist of estrogen and then the mRNA expression of adenosine receptor subtypes were evaluated. Estrogen-positive MCF-7 cells were treated with various concentrations of 17β estradiol (E2) as an estrogen agonist, and ICI 182,780 as an estrogen antagonist. The gene expression of adenosine receptor subtypes were detected by real time RT-PCR. The results of MTT assay showed that E2 increased cell viability in a dose dependent manner. The expression pattern of all adenosine receptor subtypes are as follow; A2b > A1 > A2a > A3 in untreated MCF-7 cells. Obtained results showed that E2 incubation at 0.001-0.01 μM led to up-regulation of A1ARs, A2aARs and A3ARs dose dependently. E2 at 0.001 μM also had no significant effect on A2bARs expression but, at higher doses induced a considerable decrease in mRNA A2bARs expression. Treatment with antagonist confirmed that up-regulation of these receptors is mediated by estrogen receptor. Taken together, our results indicate that treatment of MCF-7 cells with E2 led to up-regulation of adenosine receptors. However, these effects were partially restored by treatment with antagonist suggesting that such effects are mediated by estrogen receptors.

Loss of adenosine A2B receptor mediated relaxant responses in the aged female rat bladder; effects of dietary phytoestrogens

This study examined the effect of age, ovariectomy and dietary phytoestrogen ingestion on adenosine A(2B) receptor mediated relaxant responses and mRNA expression of adenosine receptor subtypes in the rat isolated bladder. Female Wistar rats (8weeks) were anaesthetised and the ovaries were removed (ovx) or left intact (sham). Rats were fed either normal rat chow (soy, phytoestrogens) or a non-soy (phytoestrogen free) diet. Isolated bladder from rats aged 12, 24 or 52weeks were pre-contracted with 3μM carbachol prior to a concentration response curve to 5'-(N-ethylcarboxamido) adenosine (NECA) being obtained. In 12-week-old rats, the bladder exhibited enhanced relaxant responses to NECA in soy-fed rats (P < 0.05), whilst at 24weeks of age, the relaxant responses to NECA were attenuated in all the groups studied except soy-treated sham rat bladders in which the relaxant responses were enhanced. At 52weeks of age, no relaxant effects were observed in any of the treatment groups and NECA-induced contractile responses occurred. In all bladders, the adenosine A(2B) receptor was the most abundantly expressed. In bladders from young and mature female rats, the mRNA expression of adenosine receptors (A(1), A(2A) and A(2B)) was lowest in the bladder from non-soy-fed ovariectomised animals and the use of phytoestrogens in the diet increased the mRNA expression of these receptors (P < 0.05). While a soy diet improves the relaxant effects to the adenosine analogue via adenosine A(2B) receptors in bladders from younger rats, the benefits are lost with advancing age.

Adenosine Can Mediate its Actions through Generation of Reactive Oxygen Species

Adenosine is an important cerebral vasodilator, but mediating mechanisms are not understood. We investigated the expression of adenosine receptor subtypes in isolated cerebral arterial muscle cells (CAMCs), and their role in adenosine-induced superoxide (O(2)(-)) generation and reduction in cerebral arterial tone. Reverse transcriptase-PCR, western blotting, and immunofluorescence studies have shown that CAMCs express transcript and protein for A1, A(2A), A(2B), and A(3) adenosine receptors. Stimulation of CAMCs with adenosine or the A(2A) agonist CGS-21680 increased the generation of O(2)(-) that was attenuated by the inhibition of A(2A) and A(2B) adenosine receptor subtypes, or by the peptide inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase gp91ds-tat, or by the mitochondria uncoupler 2,4-dinitrophenol. Application of adenosine or CGS-21680 dilated pressure-constricted cerebral arterial segments that were prevented by the antioxidants superoxide dismutase (SOD) conjugated to polyethylene glycol (PEG) and PEG-catalase or by the A(2B) adenosine receptor antagonist MRS-1754, or by the mixed A(2A) and A(2B) antagonist ZM-241385. Antagonism of the A(2A) and A(2B) adenosine receptors had no effect on cerebral vasodilatation induced by nifedipine. These findings indicate that adenosine reduces pressure-induced cerebral arterial tone through stimulation of A(2A) and A(2B) adenosine receptors and generation of O(2)(-) from NADPH oxidase and mitochondrial sources. This signaling pathway could be one of the mediators of the cerebral vasodilatory actions of adenosine.

Human Placental Adenosine Receptor Expression is Elevated in Preeclampsia and Hypoxia Increases Expression of the A 2A Receptor

Placental hypoxia as a result of impaired trophoblast invasion is suggested to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine, and the actions of adenosine are mediated through four adenosine receptors, A 1, A 2A, A 2B and A 3. We investigated the presence, distribution and expression of adenosine receptor subtypes in the human placenta, the expression of the adenosine receptors in placentas from pregnancies complicated by preeclampsia, small for gestational age (SGA) infants and uncomplicated pregnancies, and the effect of hypoxia on placental adenosine receptor expression. Immunofluorescent microscopy localized A 1, A 2A, A 2B and A 3 adenosine receptors to the syncytiotrophoblast, endothelial cells and myofibroblasts within the human placenta. Adenosine receptor protein and message expression levels were significantly higher in placentas from preeclamptic pregnancies with or without SGA infants, but not different in pregnancies with SGA infants alone. In vitro exposure of placental villous explants to hypoxia (2% oxygen) increased the expression of A 2A adenosine receptor 50%. These data indicate that all four known adenosine receptors are expressed in the human placenta and adenosine receptor expression is significantly higher in pregnancies complicated by preeclampsia. These data are consistent with the hypothesis that differences in placental adenosine receptors may contribute to alterations in placental function in preeclampsia.

Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes

To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

Differential expression of adenosine receptors in human endothelial cells: role of A2B receptors in angiogenic factor regulation.

Adenosine has been reported to stimulate or inhibit the release of angiogenic factors depending on the cell type examined. To test the hypothesis that differential expression of adenosine receptor subtypes contributes to endothelial cell heterogeneity, we studied microvascular (HMEC-1) and umbilical vein (HUVEC) human endothelial cells. Based on mRNA level and stimulation of adenylate cyclase, we found that HUVECs preferentially express A2A adenosine receptors and HMEC-1 preferentially express A2B receptors. Neither cells expressed A1 or A3 receptors. The nonselective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased expression of interleukin-8 (IL-8), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) in HMEC-1, but had no effect in HUVECs. In contrast, the selective A2A agonist 2-p-(2-carboxyethyl)phenylethylamino-NECA (CGS 21680) had no effect on expression of these angiogenic factors. Cotransfection of each type of adenosine receptors with a luciferase reporter in HMEC-1 showed that A2B receptors, but not A1, A2A, or A3, activated IL-8 and VEGF promoters. These effects were mimicked by constitutively active alphaG(q), alphaG12, and alphaG13, but not alphaG(s) or alphaG(i1-3). Furthermore, stimulation of phospholipase C indicated coupling of A2B receptors to G(q) proteins in HMEC-1. Thus, differential expression of adenosine receptor subtypes contributes to functional heterogeneity of human endothelial cells. A2B receptors, predominantly expressed in human microvascular cells, modulate expression of angiogenic factors via coupling to G(q), and possibly via G12/13.

Expression and function of adenosine receptors in human dendritic cells.

Dendritic cells (DCs) are specialized antigen-presenting cells characterized by their ability to migrate into target sites, process antigens, and activate naive T cells. In this study, we analyzed the biological activity and intracellular signaling of adenosine by using reverse transcriptase-polymerase chain reaction assays to investigate mRNA expression of A(1), A(2a) and A(3) adenosine receptors in immature and mature human DCs. Functional experiments on adenosine stimulation showed chemotaxis, intracellular calcium transients, and actin polymerization, but no activation of adenylate cyclase in immature DCs. Experiments with receptor isotype-selective agonists and antagonists as well as pertussis toxin revealed that chemotaxis, calcium transients, and actin polymerization were mediated via G(i-) or G(0-)protein-coupled A(1) and A(3) receptors. Maturation of DCs induced by lipopolysaccharide (LPS) resulted in down-regulation of A(1) and A(3) receptor mRNAs, although A(2a) receptor mRNA was still expressed. However, in LPS-differentiated DCs, adenosine and an A(2a) receptor agonist stimulated adenylate cyclase activity, enhanced intracellular cAMP levels, and inhibited interleukin 12 (IL-12) production. These effects could be completely prevented by pretreatment with A(2) receptor antagonist. These findings strongly suggest that adenosine has important but distinct biological effects in DCs activity as a chemotaxin for immature DCs and as a modulator of IL-12 production in mature DCs. These effects can be explained by differential expression of adenosine receptor subtypes.

歯肉線維芽細胞のヒアルロン酸合成酵素発現に及ぼすアデノシンの影響

Adenosine, an endogenous nucleoside, has a plethora of biological actions on a large variety of cells and can modulate the various functions of cells involved in inflammatory responses. On the other hand, production of extracellular matrices is one of the critical functions of fibroblasts. Among various extracellular matrices, hyaluronate (HA) plays important roles in migration, growth and differentiation of a variety of cells during the course of inflammatory reactions and process of wound healing.In this study, we investigated the expression of adenosine receptor subtypes in human gingival fibroblasts (HGF) and examined the effects of adenosine on the HA production of HGF by utilizing various agonists specific for adenosine receptor subtypes.Concerning the expression of adenosine receptors, RT-PCR analysis revealed that HGF expressed adenosine receptor A1, A2 a, and A2 b, but not A3 mRNA. Ligation of adenosine receptors by adenosine or adenosine analogue, 2-chlor-oadenosine (2 CADO) and N6-cyclopentyladenosine (CPA; A1 adenosine receptor agonist) but not CGS-21680 (A2 a adenosine receptor agonist) induced the expression of HA synthase mRNA, which is responsible for HA production in HGF. These results suggest that intracellular signal (s) via A1 adenosine receptor may play a central role for the upregulation of HA production by activated HGF in inflamed periodontal lesions.These results provide new evidence for the possible involvement of adenosine in the regulation of extracellular matrix production during the course of inflammatory responses in periodontal tissues.

Differential expression of adenosine A2A and A2B receptor subtypes on myeloid U937 and THP-1 cells: Adenosine A2B receptor activation selectively stimulates cAMP formation and inhibition of TNF? release in THP-1 cells

The ability of adenosine to function as an inhibitory modulator of inflammatory processes has been well documented. While the activation of adenosine A 2 receptors has been implicated in the anti-inflammatory actions of adenosine, the specific mechanisms by which adenosine modulates the function of proinflammatory cells is less well understood. The present studies were undertaken to further investigate the specific roles of adenosine A 2 receptor subtypes (A 2A and A 2B in monocyte function using two human myeloid cell lines of different developmental lineage, U937 cells (promonocytes) and THP-1 cells (monocytes). Adenosine receptor agonists stimulated the production of cAMP in both cell lines; with an apparent A 2A receptor-mediated agonist profile (CGS 21680 = NECA>> CPA) for U937 cells and an apparent A 2B receptor-mediated agonist profile (NECA>> CPA> CGS 21680) for THP-1 cells. NECA-stimulated CAMP production in both cell lines could be inhibited with the adenosine receptor antagonists CGS 15943 and DPCPX in a concentration-dependent fashion. Using RNase protection analysis, it was demonstrated that the mRNA for the adenosine A 2A receptor was expressed in U937 cells, but not in THP- 1 cells, while the adenosine A 2B receptor mRNA was detected in THP-1 cells, but not in U937 cells. Since THP-1 cells are widely used as a model of monocyte function, additional experiments were conducted to characterize the expression of adenosine receptor subtypes on these cells. Radioligand binding studies demonstrated the apparent lack of high affinity binding to A 1 , A 2A , or A 3 receptors on THP-1 cell membranes. Further, the rank order potency of adenosine receptor agonists to inhibit the LPS-stimulated release of TNFα from THP-1 cells was consistent with the activation of adenosine A 2B receptors. These results suggest the possibility that the effects of adenosine on myeloid cell function can be mediated by a differential expression of A 2 adenosine receptor subtypes during developmental maturation. Further, activation of adenosine A 2B receptors may represent a significant mechanism by which adenosine inhibits TNFα release from inflammatory cells.

Atrium- and ventricle-specific expression of adenosine receptor subtypes: Functional and molecular evidence for a stimulatory adenosine receptor subtype on ventricular myocytes

Atrium- and ventricle-specific expression of adenosine receptor subtypes: Functional and molecular evidence for a stimulatory adenosine receptor subtype on ventricular myocytes