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At least 19 recordsLinked to original sources

PubMed1998

Pharmacological characterization of adenosine A2B receptors: studies in human mast cells co-expressing A2A and A2B adenosine receptor subtypes.

Characterization of A2B receptors is hampered by the lack of selective pharmacological probes and often relies on their relative affinity to agonists that are selective at other receptor types. This approach is limited because the affinity of A2B receptors for putative A3 agonists has not been determined. Using the human erythroleukemia cell line HEL as a cellular model for A2B-mediated adenylate cyclase activation, we found the following potencies (pD2) for the non-selective agonist 5'-N-ethylcarboxamidoadenosine (NECA) (5.65 +/- 0.04), the putative A3 agonists N6-benzyl-NECA (4.17 +/- 0.06) and N6-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (IB-MECA) (3.7 +/- 0.02), and the A2A agonist 4-[(N-ethyl-5'-carbamoyladenos-2-yl)-aminoethyl]-phenylpropionic acid (CGS21680) (2.8 +/- 0.1). Because of the lack of a selective agonist, characterization of A2B receptor function is difficult in cells co-expressing A2A receptors. In the human mast cell line HMC-1, NECA induced cAMP accumulation with a concentration-response relationship best fitted to a two-sited model (pD2 7.69 +/- 0.42 and 5.92 +/- 0.21 for high- and low-affinity sites), suggesting the presence of both A2A and A2B receptors in these cells. We demonstrated that A2B receptors can be selectively activated with NECA in the presence of the selective A2A antagonist 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c ]pyrimidine (SCH 58261). Under these conditions, the concentration-response relationship of NECA for cyclic AMP accumulation was now best fitted to a one-site model (pD2 5.68 +/- 0.03, Hill slope 0.93 +/- 0.06, 95% confidence intervals 0.8 to 1.06) corresponding to selective activation of A2B receptors. Using the approaches developed in this study, we determined that A2B, and not A2A or A3, receptors account for all the calcium mobilization induced by NECA in HMC-1 cells.

Adenosine↗

PubMed1999

Use of the triazolotriazine [3H]ZM 241385 as a radioligand at recombinant human A2B adenosine receptors.

Radiolabeled ZM 241385 (4-(2-[7-amino-2- ¿furyl¿¿1,2,4¿triazolo¿2,3-a¿¿1,3,5¿triazin-5-ylaminoethyl)p henol), has previously been used as a high affinity radioligand for the labeling of A2A adenosine receptors in cell membranes. Another subtype, the A2B receptor, is the least well-defined subtype of adenosine receptors and lacks selective pharmacological probes. In the present study, we have used [3H]ZM 241385 as a radioligand to label recombinant human A2B adenosine receptors in HEK-293 cell membranes, that do not express A2A adenosine receptors, and found that the pharmacological profile is consistent with the SAR of A2B receptors. Saturable, specific binding (Kd 33.6 nM, Bmax 4.48 pmol/mg protein) that was best described by a one-site model was found, and specific binding was approximately 75% of total binding. [3H]ZM 241385 binding was displaceable by a large number of compounds known to interact with A2B receptors; thus, this method has promise as a tool in the search for agonists and antagonists selective for this subtype. Xanthine analogs, which are antagonists, proved to be the most potent displacers. The Ki of XAC, xanthine amine congener, was 12.3 nM, while CPX (8-cyclopentyl-1,3-dipropylxanthine) was less potent. The non-selective triazoloquinazoline antagonist CGS 15943 (Ki 16.4 nM), which is similar in structure to ZM 241385, was slightly less potent than XAC. The non-xanthine A2B-antagonist alloxazine displaced [3H]ZM 241385-binding with a Ki of 462 nM, similar to its affinity in functional assays. Adenosine derivatives known to activate this receptor subtype, such as NECA (5'-N-ethylcarboxamidoadenosine) and R-PIA (N6-phenylisopropyladenosine), were considerably less potent than the 8-substituted xanthines examined.

Animals↗

PubMed2002

Structure-activity relationships at human and rat A2B adenosine receptors of xanthine derivatives substituted at the 1-, 3-, 7-, and 8-positions.

In the search for improved selective antagonist ligands of the A2B adenosine receptor, which have the potential as antiasthmatic or antidiabetic drugs, we have synthesized and screened a variety of alkylxanthine derivatives substituted at the 1-, 3-, 7-, and 8-positions. Competition for 125I-ABOPX (125I-3-(4-amino-3-iodobenzyl)-8-(phenyl-4-oxyacetate)-1-propylxanthine) binding in membranes of stably transfected HEK-293 cells revealed uniformly higher affinity (<10-fold) of these xanthines for human than for rat A2B adenosine receptors. Binding to rat brain membranes expressing A1 and A2A adenosine receptors revealed greater A2B selectivity over A2A than A1 receptors. Substitution at the 1-position with 2-phenylethyl (or alkyl/olefinic groups) and at N-3 with hydrogen or methyl favored A2B selectivity. Relative to enprofylline 2b, pentoxifylline 35 was equipotent and 1-propylxanthine 3 was >13-fold more potent at human A2B receptors. Most N-7 substituents did not enhance affinity over hydrogen, except for 7-(2-chloroethyl), which enhanced the affinity of theophylline by 6.5-fold to 800 nM. The A2B receptor affinity-enhancing effects of 7-(2-chloroethyl) vs 7-methyl were comparable to the known enhancement produced by an 8-aryl substitution. Among 8-phenyl analogues, a larger alkyl group at the 1-position than at the 3-position favored affinity at the human A2B receptor, as indicated by 1-allyl-3-methyl-8-phenylxanthine, with a K(i) value of 37 nM. Substitution on the 8-phenyl ring indicated that an electron-rich ring was preferred for A2B receptor binding. In conclusion, new leads for the design of xanthines substituted in the 1-, 3-, 7-, and 8-positions as A2B receptor-selective antagonists have been identified.

Animals↗

PubMed1999

IFN-gamma up-regulates the A2B adenosine receptor expression in macrophages: a mechanism of macrophage deactivation.

Adenosine is a potent endogenous anti-inflammatory agent released by cells in metabolically unfavorable conditions, such as hypoxia or ischemia. Adenosine modulates different functional activities in macrophages. Some of these activities are believed to be induced through the uptake of adenosine into the macrophages, while others are due to the interaction with specific cell surface receptors. In murine bone marrow-derived macrophages, the use of different radioligands for adenosine receptors suggests the presence of A2B and A3 adenosine receptor subtypes. The presence of A2B receptors was confirmed by flow cytometry using specific Abs. The A2B receptor is functional in murine macrophages, as indicated by the fact that agonists of A2B receptors, but not agonists for A1, A2A, or A3, lead to an increase in cAMP levels. IFN-gamma up-regulates the surface protein and gene expression of the A2B adenosine receptor by induction of de novo synthesis. The up-regulation of A2B receptors correlates with an increase in cAMP production in macrophages treated with adenosine receptor agonist. The stimulation of A2B receptors by adenosine or its analogues inhibits the IFN-gamma-induced expression of MHC class II genes and also the IFN-gamma-induced expression of nitric oxide synthase and of proinflammatory cytokines. Therefore, the up-regulation of the A2B adenosine receptor expression induced by IFN-gamma could be a feedback mechanism for macrophage deactivation.

Adenosine↗

PubMed1998

Agonist-induced desensitization of A2B adenosine receptors.

Agonist-induced desensitization has been described for the A1, A2A, and A3 adenosine receptor subtypes of the G protein-coupled receptor superfamily. Desensitization of the fourth adenosine receptor subtype, the A2B adenosine receptor (A(2B)R), has not been studied extensively. We sought to determine whether the A(2B)R is subject to agonist-induced desensitization. COS 7 cells, which exhibit endogenous expression of the A(2B)R, and transfected CHO cells, which stably express a modified rat A(2B)R bearing a 5' FLAG epitope tag, were studied. Cyclic AMP (cAMP) responsiveness to an acute challenge was measured after pretreating (desensitizing) cells with the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA). Incubation with NECA resulted in hyporesponsiveness to acute agonist challenge in both COS 7 and transfected CHO cells. Desensitized cells exhibited restoration of cAMP responses after recovery for 24 hr in growth medium. Choleratoxin-induced cAMP responses were preserved in desensitized cells, and high concentrations of NECA were unable to overcome the desensitization. Membrane levels of the epitope-tagged A(2B)R were assessed by western blot in transiently transfected COS 7 cells. The expression of epitope-tagged A(2B)Rs was not different between control and desensitized cells. In northern blot analysis, levels of endogenous A(2B)R mRNA were similar in control and desensitized COS 7 cells. We conclude that the A(2B)R is subject to agonist-induced desensitization with preserved expression of A(2B)R mRNA and protein. Uncoupling of the A2B adenosine receptor from the G protein complex may contribute to the mechanism of desensitization.

Adenosine-5'-(N-ethylcarboxamide)↗

PubMed2007

Enhanced mast cell activation in mice deficient in the A2b adenosine receptor.

Antigen-mediated cross-linking of IgE bound to mast cells via the high affinity receptor for IgE triggers a signaling cascade that results in the release of intracellular calcium stores, followed by an influx of extracellular calcium. The collective increase in intracellular calcium is critical to the release of the granular contents of the mast cell, which include the mediators of acute anaphylaxis. We show that the sensitivity of the mast cell to antigen-mediated degranulation through this pathway can be dramatically influenced by the A2b adenosine receptor. Loss of this Gs-coupled receptor on mouse bone marrow-derived mast cells results in decreased basal levels of cyclic AMP and an excessive influx of extracellular calcium through store-operated calcium channels following antigen activation. Mice lacking the A2b receptor display increased sensitivity to IgE-mediated anaphylaxis. Collectively, these findings show that the A2b adenosine receptor functions as a critical regulator of signaling pathways within the mast cell, which act in concert to limit the magnitude of mast cell responsiveness when antigen is encountered.

Anaphylaxis↗

PubMed1997

An endogenous A2B adenosine receptor coupled to cyclic AMP generation in human embryonic kidney (HEK 293) cells.

1. Cyclic AMP generation by adenosine analogues was examined in human embryonic kidney (HEK 293) cells by use of a [3H]-adenine pre-labelling methodology. 2. Adenosine analogues showed the following rank order of potency (pD2 value): 5'-N-ethylcarboxamidoadenosine (NECA, 5.24)>2-chloroadenosine (4.41) > or = adenosine (4.19)= N6-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.11). The A2A-selective agonist CGS21680 failed to elicit a significant stimulation of cyclic AMP generation at concentrations below 30 microM. 3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity, while in comparison maximal responses to adenosine (76+/-8% NECA response), 2-chloroadenosine (70+/-6%) and APNEA (40+/-3%) were significantly reduced. 4. Antagonists of the NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA2 value): CGS 15943 (7.79)=XAC (7.74)>DPCPX (7.01)=PD115199 (6.93) 8FB-PTP (6.80)>KF 17837 (5.98)>3-propylxanthine (5.13). 5. Agarose gel electrophoresis of the products of the polymerase chain reaction, with cDNA generated from HEK 293 cell total RNA showed virtually identical patterns and nucleotide sizes in comparison with the vector for the full length human brain A2B adenosine receptor. 6. We concluded that HEK 293 cells express an endogenous adenosine receptor coupled to cyclic AMP generation which is of the A2B subtype.

2-Chloroadenosine↗

PubMed1999

Expression of A2B adenosine receptors in human lymphocytes: their role in T cell activation.

Extracellular adenosine has a key role in the development and function of the cells of the immune system. Many of the adenosine actions seem to be mediated by specific surface receptors positively coupled to adenylate cyclase: A2A and A2B. Despite the fact that A2A receptors (A2ARs) can be easily studied due to the availability of the specific agonist CGS21680, a pharmacological and physiological characterization of adenosine A2B receptors (A2BRs) in lymphocytes has not been possible due to the lack of suitable reagents. Here we report the generation and characterization of a polyclonal antipeptide antibody raised against the third extracellular loop of the A2BR human clone which is useful for immunocytochemical studies. This antibody has permitted the detection of A2BR+ cells in lymphocyte samples isolated from human peripheral blood. The pharmacology of cAMP-producing compounds is consistent with the presence of functional A2BRs but not of A2A receptors in these human cells. The percentage of A2BR-expressing cells was similar in the CD4(+) or CD8(+) T cell subpopulations. Interestingly activation signals delivered by either phytohemagglutinin or anti-T cell receptor/CD3 complex antibodies led to a significant increase in both the percentage of cells expressing the receptor and the intensity of the labeling. These receptors are functional since interleukin-2 production in these cells is reduced by NECA but not by R-PIA or CGS21680. These results show that A2BR expression is regulated in T cell activation and suggest that the role of adenosine in lymphocyte deactivation is mediated by A2BRs.

Adenosine↗

PubMed2004

A2B adenosine receptor activity is reduced in neutrophils from patients with systemic sclerosis.

We conducted the present study to investigate protein expression and functioning of A2A and A2B adenosine receptors (ARs) in neutrophils of patients affected by systemic sclerosis (SSc). The presence of A2A and A2B ARs was assessed by immunoblotting using specific antibodies. Equilibrium A2A and A2B ARs binding parameters were evaluated by radioligand binding assay. Functional studies were conducted to investigate coupling of the A2B AR to the adenylyl cyclase pathway. This is the first report of the use of Western blot analysis to confirm the presence of A2A and A2B ARs in human neutrophils. No significant changes in A2A AR binding parameters or expression levels were detected between SSc patients and healthy control individuals. A significant decrease (65%) in the maximum density of A2B AR binding sites occurred in SSc neutrophils, whereas no changes in the affinity constant values were found. Moreover, a decrease in A2B AR mediated adenylyl cyclase activity was observed in patients with SSc. Our findings demonstrate the occurrence of selective alterations in A2B AR density and signalling in SSc.

Adenosine↗

PubMed2001

Adenosine A2B receptors behave as an alternative anchoring protein for cell surface adenosine deaminase in lymphocytes and cultured cells.

Adenosine deaminase (ADA) is an enzyme of the purine metabolism that has been largely considered to be cytosolic. Recently, it has been demonstrated that the enzyme appears on the surface of lymphocytes where it interacts with the T-cell activation antigen CD26. ADA also appears on the surface of nonlymphoid cells anchored to adenosine A1 receptors. Here it is demonstrated that cell surface ADA in ADA+/CD26- T lymphocytes anchors to adenosine receptors of the A2B subtype (A2BR). An interaction between A2BR and cell surface ADA has been demonstrated in transfected Chinese hamster ovary cells and Jurkat J32 T lymphocytes. This has been proved by coimmunoprecipitation, binding of exogenous ADA to A2BR+ cells, and coimmunolocalization. The specificity of the interaction has also been demonstrated by the lack of interaction with other members of the G protein-coupled receptor superfamily. Binding of ADA to A2BR increases the affinity of the agonist 5'-N-ethylcarboxamidoadenosine and cAMP production. This effect occurs even when ADA devoid of enzyme activity is used. Therefore, in lymphocytes, cell surface ADA, apart from degrading extracellular adenosine, regulates those actions of adenosine that are mediated via adenosine receptors of the A2B subtype.

Adenosine Deaminase↗

PubMed1999

Evidence for A1 and A2B adenosine receptors in baby hamster kidney (BHK) cells.

Adenosine is known to produce biphasic effects in the renal tissues via adenosine receptors. However, the presence of more than one subtype of adenosine receptor on a type of kidney cell or tissue has not been conclusively demonstrated. To address this issue, we investigated the presence of A1 and A2 adenosine receptors in baby hamster kidney (BHK) cells by use of radioligand binding and the reverse transcription-polymerase chain reaction. Ligand binding studies with (3H)-DPCPX revealed a single class of binding site with a K(D) of 9.2 +/- 2.0 nM, a Bmax of 1.7 +/- 0.2 pmol/mg protein and a pharmacological profile characteristic of A1 adenosine receptor on the BHK cell membrane. As the presence of A2 adenosine receptors could not be conclusively determined by ligand binding studies, the more sensitive method of RT-PCR was employed. The presence of A1 and A2B adenosine receptors was detected by RT-PCR with specific primers and the subsequent sequencing of the resultant amplification product. The sequences obtained were 75-90% homologous to the respective adenosine receptor mRNA of rat, mouse and human.

Adenosine-5'-(N-ethylcarboxamide)↗

PubMed1999

Altered gene expression in melanocytes exposed to 4-tertiary butyl phenol (4-TBP): upregulation of the A2b adenosine receptor 1.

Exposure to phenolic agents contributes to the development of occupational vitiligo. Proposed as a causative factor for leukoderma in vivo, the para-substituted phenol 4-tertiary butyl phenol was chosen to investigate early cellular events responsible for selective disappearance of melanocytes from the epidermis of individuals sensitive to such agents. To this end, differential display of melanocyte mRNA isolated from three separate cultures was performed following a 12 h exposure of cells to 250 microM 4-tertiary butyl phenol or to vehicle alone. Fragments of cDNA representing differentially expressed messages were cloned and subsequently confirmed by reverse dot blotting. Alignment analysis revealed that the L30 ribosomal protein was upregulated by the treatment, potentially reflecting altered levels of protein synthesis in response to stress. In addition, a gene sequence upregulated following exposure to 4-tertiary butyl phenol was identified as the A2b receptor (a P1 receptor for adenosine). Differential expression of this gene was confirmed in an RNase protection assay. By reverse transcription-polymerase chain reaction, the gene was shown to be expressed in keratinocytes and fibroblasts as well. Flow cytometry confirmed differential expression in melanocytes and fibroblasts, but not in keratinocytes. Interestingly, it has been reported that P1 purinoceptor stimulation can induce apoptosis. This is in concordance with results reported elsewhere demonstrating induction of apoptosis by 4-tertiary butyl phenol in human melanocytes, as well as with morphologic changes observed in this study in cells exposed to 250 microM 4-tertiary butyl phenol for 72 h. In conclusion, differential display is useful to establish melanocyte components involved in the cellular response to phenolic agents.

Flow Cytometry↗

PubMed2004

Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells.

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.

Adenosine↗

PubMed1998

Adenosine A2B receptors: a novel therapeutic target in asthma?

Adenosine is an endogenous nucleoside that modulates many physiological processes. Its actions are mediated by interaction with specific cell membrane receptors. Four subtypes of adenosine receptor have been cloned: A1, A2A, A2B and A3. Significant advancement has been made in our understanding of the molecular pharmacology and physiological relevance of adenosine receptors but our knowledge of A2B receptors lags behind that of other receptor types. Only recently have potentially important functions been discovered for the A2B receptors, prompting a renewed interest in this receptor type. A2B receptors have been implicated in the regulation of vascular smooth muscle tone, cell growth, intestinal function and neurosecretion. In this review, Igor Feoktistov, Riccardo Polosa, Stephen Holgate and Italo Biaggioni focus on the role of A2B receptors in mast cell activation and the potential relevance of this action on asthma.

Anti-Asthmatic Agents↗

PubMed2000

Polarity of A2b adenosine receptor expression determines characteristics of receptor desensitization.

UNLABELLED: It is not known if, in polarized cells, desensitization events can be influenced by the domain on which the receptor resides. Desensitization was induced by 5'-(N-ethylcarboxamido)adenosine (NECA) and was quantitated by measurement of short-circuit current (I(sc)) in response to adenosine. NECA added to either the apical or basolateral compartments rapidly desensitized receptors on these respective domains. Although apical NECA had no effect on the basolateral receptor stimulation, basolateral NECA induced a complete desensitization of the apical receptor. We hypothesized that desensitization of apical receptor by basolateral desensitization could relate to a trafficking step in which A2b receptor is first targeted basolaterally upon synthesis and transported to the apical surface via vesicular transport/microtubules. Because desensitization is associated with downregulation of receptors, apical adenosine receptor can thus be affected by basolateral desensitization. Both low temperature and nocodazole inhibited I(sc) induced by apical and not basolateral adenosine. IN CONCLUSION: 1) a single receptor subtype, here modeled by the A2b receptor, differentially desensitizes based on the membrane domain on which it is expressed, 2) agonist exposure on one domain can result in desensitization of receptors on the opposite domain, 3) cross-domain desensitization can display strict polarity, and 4) receptor trafficking may play a role in the cross-desensitization process.

Adenosine↗

PubMed1998

A functional screening of adenosine analogues at the adenosine A2B receptor: a search for potent agonists.

Various adenosine analogues were tested at the adenosine A2B receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A2B receptors. 5'-N-Substituted carboxamidoadenosines were most potent. 5'-N-Ethylcarboxamidoadenosine (NECA) was most active with an EC50 value of 3.1 microM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N6) of the purine ring system. The most active N6-substituted derivative N6-methyl-NECA was 5 fold less potent than NECA. C8- and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3- and 7-deazaanalogues were not active.

Adenosine↗

PubMed2003

Reduction in preretinal neovascularization by ribozymes that cleave the A2B adenosine receptor mRNA.

Adenosine modulates a variety of cellular functions by interacting with specific cell surface G protein-coupled receptors (A1, A2A, A2B, and A3) and is a potential mediator of angiogenesis through the A2B receptor. The lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our goal was to design a hammerhead ribozyme that would specifically cleave the A2B receptor mRNA and examine its effect on retinal angiogenesis. Ribozymes specific for the mouse and human A2B receptor mRNAs were designed and cloned in expression plasmids. Human embryonic kidney (HEK) 293 cells were transfected with these plasmids and A2B receptor mRNA levels were determined by quantitative real-time RT-PCR. Human retinal endothelial cells (HRECs) were also transfected and cell migration was examined. The effects of these ribozymes on the levels of preretinal neovascularization were determined using a neonatal mouse model of oxygen-induced retinopathy (OIR). We produced a ribozyme with a Vmax of 515+/-125 pmol/min and a Kcat of 36.1+/-8.3 min(-1) (P< or =1x10(-5)). Transfection of HEK293 cells with the plasmid expressing the ribozyme reduced A2B receptor mRNA levels by 45+/-4.8% (P=5.1x10(-5)). Transfection of HRECs reduced NECA-stimulated migration of cells by 47.3+/-1.2% (P=7x10(-4)). Intraocular injection of the constructs into the mouse model reduced preretinal neovascularization by 53.5+/-8.2% (P=4.5x10(-5)). Our results suggest that the A2B receptor ribozyme will provide a tool for the selective inhibition of this receptor and provide further support for the role of A2B receptor in retinal angiogenesis.

Animals↗

PubMed2001

Inhibition of NF-kappaB-mediated gene transcription by the human A2B adenosine receptor in Chinese hamster ovary cells.

NF-kappaB is a transcription factor that plays a vital role in regulating inducible gene expression in immune and inflammatory responses. In view of the well documented effects of adenosine on immune and inflammatory responses, we have explored whether adenosine A1, A2B and A3 receptors regulate NF-kappaB activity in transfected Chinese hamster ovary (CHO) cells using a luciferase reporter gene construct. No increases in NF-kappaB activity were observed in CHO-A1, -A2B and -A3 cells stimulated with the non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine. Elevating intracellular cyclic AMP (cAMP) levels using forskolin (direct activator of adenylyl cyclase) and rolipram (type IV, cAMP-specific phosphodiesterase inhibitor), inhibited NF-kappaB activity in CHO cells. Adenosine A2B receptor stimulation also inhibited NF-kappaB activity, whereas adenosine A1 and A3 receptor activation had no effect. These data reflect the known coupling of adenosine A2B receptors to increases in cAMP. In conclusion, adenosine A1, A2B and A3 receptors do not directly activate NF-kappaB in CHO cells. However, adenosine A2B receptor activation significantly inhibited NF-kappaB activity. Inhibition of NF-kappaB activity by the adenosine A2B receptor may contribute to the anti-inflammatory effects of adenosine.

Analysis of Variance↗