A2B Receptor Activation Research Articles

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.

Switching Sides to Get Ahead

As the nervous system of the developing vertebrate embryo is laid down, the guidance molecule netrin attracts the growth cones of advancing axons to the embryo's midline. However, as soon as the growth cones cross the midline, they switch allegiance, ignoring netrin and instead responding to Slit, which repels the growth cones away from the midline. Two papers tackle how growth cones make their way through competing signals. Stein and Tessier-Lavigne show that this switch is the result of the cytoplasmic domain of the Slit receptor, Robo, binding to the cytoplasmic domain of the netrin receptor DCC. Thus, the dwindling effects of netrin on growth cones are directly linked to their increasing responsiveness to the repellent effects of Slit. This interlocked silencing mechanism prevents the growth cone from being trapped in a tug-of-war between the attractive and repellent activities of two opposing guidance molecules. Stein et al. address the mechanism by which netrin exerts its effects. In contrast to a recent suggestion that netrin acts through the adenosine A2B receptor, the authors show that netrin binds directly to the DCC protein and that A2B activation is not required for netrin's effects on axon outgrowth or neuron attraction. Thus, DCC is the primary receptor responsible for netrin's actions. Dickson provides a Perspective on axon guidance. E. Stein, M. Tessier-Lavigne, Hierarchical organization of guidance receptors: Silencing of netrin attraction by Slit through a Robo/DCC receptor complex. Science 291 , 1928-1938 (2001). [Abstract] [Full Text] E. Stein, Y. Zou, M.-m. Poo, M. Tessier-Lavigne, Binding of DCC by netrin-1 to mediate axon guidance independent of adenosine A2B receptor activation. Science 291 , 1976-1982 (2001). [Abstract] [Full Text] B. J. Dickson, Moving on. Science 291 , 1910-1911 (2001). [Full Text]

Binding of DCC by netrin-1 to mediate axon guidance independent of adenosine A2B receptor activation.

Netrins stimulate and orient axon growth through a mechanism requiring receptors of the DCC family. It has been unclear, however, whether DCC proteins are involved directly in signaling or are mere accessory proteins in a receptor complex. Further, although netrins bind cells expressing DCC, direct binding to DCC has not been demonstrated. Here we show that netrin-1 binds DCC and that the DCC cytoplasmic domain fused to a heterologous receptor ectodomain can mediate guidance through a mechanism involving derepression of cytoplasmic domain multimerization. Activation of the adenosine A2B receptor, proposed to contribute to netrin effects on axons, is not required for rat commissural axon outgrowth or Xenopus spinal axon attraction to netrin-1. Thus, DCC plays a central role in netrin signaling of axon growth and guidance independent of A2B receptor activation.

A(2B) receptors mediate antimitogenesis in vascular smooth muscle cells.

Adenosine inhibits growth of vascular smooth muscle cells. The goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat aortic vascular smooth muscle cells, platelet-derived growth factor-BB (PDGF-BB) (25 ng/mL) stimulated DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell number), collagen synthesis ([(3)H]proline incorporation), total protein synthesis ([(3)H]leucine incorporation), and mitogen-activated protein (MAP) kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, but not N(6)-cyclopentyladenosine or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A(2B) receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1, 3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A(2) receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A(2B) receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and MAP kinase activity. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5'-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A(2B) receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of vascular smooth muscle cell growth by activating A(2B) receptors coupled to inhibition of MAP kinase activity. Pharmacological or molecular biological activation of A(2B) receptors may prevent vascular remodeling associated with hypertension, atherosclerosis, and restenosis following balloon angioplasty.

Glucose and Glutamine Gavage Increase Portal Vein Nitric Oxide Metabolite Levels via Adenosine A2b Activation

Introduction.Postprandial intestinal hyperemia is a complex vascular response during nutrient absorption. Many mediators have been studied including enteric reflexes, GI hormones, and absorption-stimulated metabolic mediators such as pH and adenosine. We have shown that nitric oxide (NO) mediates premucosal arteriolar dilation during glucose absorption and that glucose-induced portal vein NO metabolite production requires adenosine A2b receptor activation. We hypothesize that Na+-linked absorption ofl-glutamine orl-glycine might also stimulate NO release in the enteroportal circulation via adenosine A2b receptors.Methods.Male Sprague–Dawley rats (190–220 g) were anesthetized with urethane/α-chloralose and cannulated for hemodynamic monitoring and blood sampling. A right paramedian abdominal incision was made for access to both the stomach (gavage) and the portal vein (blood sampling). Animals received intragastric nutrient gavage (saline,d-glucose,l-glutamine, racemic glycine, or oleic acid) with and without adenosine A2b receptor blockade. NO metabolites (NOx) were measured by a fluorescent modified-Greiss assay at baseline and 30 min after nutrient gavage.Results.Glucose and glutamine gavage increased portal NOxlevels compared to baseline, while glycine and oleic acid gavage did not. Adenosine A2b antagonism returned NOxlevels to baseline in both glucose and glutamine gavage animals, but did not alter portal NOxlevels in glycine- or oleic acid-treated animals.Conclusions.These data suggest that nutrient-induced adenosine is involved in a signaling process from the intestinal epithelium to nitric oxide-producing cells elsewhere in the vasculature. Adenosine A2b receptors are required for NO production during Na+-linked glucose or glutamine absorption.

A2B adenosine receptors mediate relaxation of the pig intravesical ureter: adenosine modulation of non adrenergic non cholinergic excitatory neurotransmission.

1. The present study was designed to characterize the adenosine receptors involved in the relaxation of the pig intravesical ureter, and to investigate the action of adenosine on the non adrenergic non cholinergic (NANC) excitatory ureteral neurotransmission. 2. In U46619 (10(-7) M)-contracted strips treated with the adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI, 10(-6) M), adenosine and related analogues induced relaxations with the following potency order: 5'-N-ethylcarboxamidoadenosine (NECA) = 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA) = 2-chloroadenosine (2-CA) > adenosine > cyclopentyladenosine (CPA) = N6-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) = 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoaden os ine (CGS21680). 3. Epithelium removal or incubation with indomethacin (3 x 10(-6) M) and L-N(G)-nitroarginine (L-NOARG, 3 x 10(-5) M), inhibitors of prostanoids and nitric oxide (NO) synthase, respectively, failed to modify the relaxations to adenosine. 4. 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10(-8) M) and 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 3 x 10(-8) M and 10(-7) M), A1 and A2A receptor selective antagonists, respectively, did not modify the relaxations to adenosine or NECA. 8-phenyltheophylline (8-PT, 10(-5) M) and DPCPX (10(-6) M), which block A1/A2-receptors, reduced such relaxations. 5. In strips treated with guanethidine (10(-5) M), atropine (10(-7) M), L-NOARG (3 x 10(-5) M) and indomethacin (3 x 10(-6) M), both electrical field stimulation (EFS, 5 Hz) and exogenous ATP (10(-4) M) induced contractions of preparations. 8-PT (10(-5) M) increased both contractions. DPCPX (10(-8) M), NECA (10(-4) M), CPCA, (10(-4) M) and 2-CA (10(-4) M) did not alter the contractions to EFS. 6. The present results suggest that adenosine relaxes the pig intravesical ureter, independently of prostanoids or NO, through activation of A2B-receptors located in the smooth muscle. This relaxation may modulate the ureteral NANC excitatory neurotransmission through a postsynaptic mechanism.

Neutrophil-derived 5'-adenosine monophosphate promotes endothelial barrier function via CD73-mediated conversion to adenosine and endothelial A2B receptor activation.

During episodes of inflammation, polymorphonuclear leukocyte (PMN) transendothelial migration has the potential to disturb vascular barrier function and give rise to intravascular fluid extravasation and edema. However, little is known regarding innate mechanisms that dampen fluid loss during PMN-endothelial interactions. Using an in vitro endothelial paracellular permeability model, we observed a PMN-mediated decrease in endothelial paracellular permeability. A similar decrease was elicited by cell-free supernatants from activated PMN (FMLP 10(-6) M), suggesting the presence of a PMN-derived soluble mediator(s). Biophysical and biochemical analysis of PMN supernatants revealed a role for PMN-derived 5'-adenosine monophosphate (AMP) and its metabolite, adenosine, in modulation of endothelial paracellular permeability. Supernatants from activated PMN contained micromolar concentrations of bioactive 5'-AMP and adenosine. Furthermore, exposure of endothelial monolayers to authentic 5'-AMP and adenosine increased endothelial barrier function more than twofold in both human umbilical vein endothelial cells and human microvascular endothelial cells. 5'-AMP bioactivity required endothelial CD73-mediated conversion of 5'-AMP to adenosine via its 5'-ectonucleotidase activity. Decreased endothelial paracellular permeability occurred through adenosine A2B receptor activation and was accompanied by a parallel increase in intracellular cAMP. We conclude that activated PMN release soluble mediators, such as 5'-AMP and adenosine, that promote endothelial barrier function. During inflammation, this pathway may limit potentially deleterious increases in endothelial paracellular permeability and could serve as a basic mechanism of endothelial resealing during PMN transendothelial migration.

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.

Adenosine inhibits collagen and protein synthesis in cardiac fibroblasts: role of A2B receptors.

The objective of this study was to characterize the effects of exogenous and endogenous (cardiac fibroblast-derived) adenosine on [3H]proline and [3H]leucine incorporation, which are reliable markers of collagen and total protein synthesis, respectively, in rat left ventricular cardiac fibroblasts. Growth-arrested confluent cardiac fibroblast monolayers were stimulated with 2.5% fetal calf serum (FCS) in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), or modulators of adenosine levels including (1) erythro-9-(2-hydroxy-3-nonyl) adenine (adenosine deaminase inhibitor), (2) dipyridamole (adenosine transport blocker), and (3) iodotubericidin (adenosine kinase inhibitor). All agents inhibited in a concentration-dependent fashion FCS-induced [3H]proline and [3H]leucine incorporation. These effects were blocked by KF17837 (selective A2 antagonist) and 1,3-dipropyl-8-(p-sulfophenyl)xanthine (A1/A2 receptor antagonist) but not by 8-cyclopentyl-1,3-dipropylxanthine (selective A1 antagonist), thus excluding the participation of A1 receptors. The lack of effect of CGS21680 (selective A2A agonist) excluded involvement of A2A receptors, thus suggesting a major role for A2B receptors. Comparisons of the inhibitory potencies of N6-cyclopentyladenosine (selective A1 agonist), 5'-N-ethylcarboxamidoadenosine (A1/A2 agonist), and 5'-N-methylcarboxamidoadenosine (A1/A2 agonist) were consistent with that of an A2B receptor subtype mediating the inhibitory effects. We conclude that adenosine inhibits FCS-induced collagen and total protein synthesis in cardiac fibroblasts via activation of A2B receptors. These studies suggest, but do not prove, that endogenous adenosine may protect against cardiac fibrosis.

Adenosine inhibits growth of human aortic smooth muscle cells via A2B receptors.

Adenosine inhibits rat vascular smooth muscle cell (SMC) growth. However, the effects of adenosine on human vascular SMC proliferation and synthesis of extracellular matrix proteins, such as collagen, are unknown. The objective of this study was to characterize the effects of exogenous and endogenous (SMC-derived) adenosine on human aortic SMC proliferation and collagen synthesis. Growth-arrested SMCs were stimulated with 2.5% fetal calf serum (FCS) in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), and with agents that increase endogenous adenosine levels, including erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), dipyridamole, and iodotubericidin. All of these agents inhibited in a concentration-dependent manner FCS-induced SMC proliferation as assessed by DNA synthesis (3H-thymidine incorporation) and cell counting, as well as collagen synthesis (3H-proline incorporation). EHNA, dipyridamole, and iodotubericidin increased extracellular levels of adenosine by 1.7-fold to 18-fold when added separately to SMCs, and EHNA+iodotubericidin and EHNA+iodotubericidin+dipyridamole increased extracellular adenosine levels by more than 392-fold. Both KF17837 (selective A2 antagonist) and DPSPX (A1/A2 antagonist), but not DPCPX (selective A1 antagonist), blocked the antimitogenic effects of 2-chloroadenosine, EHNA, and dipyridamole on DNA and collagen synthesis, suggesting the involvement of A2A and/or A2B, but excluding the participation of A1, receptors. The lack of effect of CGS21680 (selective A2A agonist), excluded involvement of A2A receptors and suggested a major role for A2B receptors. A comparison of the inhibitory potencies of 2-chloroadenosine, N6-cyclopentyladenosine (selective A1 agonist), NECA (A1/A2 agonist), and MECA (A1/A2 agonist) were consistent with an A2B receptor subtype mediating the inhibitory effects of adenosine on human aortic SMC proliferation. In conclusion, human aortic SMCs synthesize adenosine, and exogenous as well as endogenous (SMC-derived) adenosine inhibits SMC proliferation and collagen synthesis via activation of A2B receptors.

Calcium mobilization in Jurkat cells via A2b adenosine receptors.

1. A functional study of cell surface A2b adenosine receptors was performed on the T cell leukaemia line, Jurkat. 2. A2b receptors were coupled both to the adenylate cyclase system and to intracellular calcium channels. In fact, the agonist of A2b receptors, 5'-N-ethylcarboxamidoadenosine (NECA), led to a transient accumulation of intracellular calcium by an inositol phosphate-independent mechanism. 3. The NECA-induced accumulation of cGMP was not responsible for the calcium mobilization via A2b receptors. 4. The calcium response elicited by activation of A2b receptors was independent of that evoked by activation of the T cell receptor. 5. These findings not only delineate a novel transduction mechanism for adenosine but also support a specific role for adenosine in modulating signals elicited via the T cell receptor.

Exogenous and endogenous adenosine inhibits fetal calf serum-induced growth of rat cardiac fibroblasts: role of A2B receptors.

Because proliferation of cardiac fibroblasts participates in cardiac hypertrophy/remodeling associated with hypertension and myocardial infarction, it is important to elucidate factors regulating cardiac fibroblast proliferation. Adenosine, a nucleoside abundantly produced by cardiac cells, is antimitogenic vis-à-vis vascular smooth muscle cells; however, the effect of adenosine on cardiac fibroblast proliferation is unknown. The objective of this study was to characterize the effects of exogenous and endogenous (cardiac fibroblast-derived) adenosine on cardiac fibroblast proliferation. Growth-arrested cardiac fibroblasts were stimulated with 2.5% FCS in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), or modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; adenosine deaminase inhibitor); (2) dipyridamole (adenosine transport blocker); and (3) iodotubericidin (adenosine kinase inhibitor). All of these agents inhibited, in a concentration-dependent manner, FCS-induced cardiac fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation) and cell counting. EHNA, dipyridamole, and iodotubericidin increased extracellular levels of adenosine by 2.3- to 5.6-fold when added separately to cardiac fibroblasts, and EHNA+iodotubericidin or EHNA+iodotubericidin+dipyridamole increased extracellular adenosine levels by >690-fold. Both KF17837 (selective A2 antagonist) and DPSPX (nonselective A2 antagonist) but not DPCPX (selective A1 antagonist) blocked the antimitogenic effects of 2-chloroadenosine, EHNA, and dipyridamole on DNA synthesis, suggesting the involvement of A2A and/or A2B but excluding the participation of A1 receptors. The lack of effect of CGS21680 (selective A2A agonist) excluded involvement of A2A receptors and suggested a major role for A2B receptors. This conclusion was confirmed by the rank order potencies of four adenosine analogues. Cardiac fibroblasts synthesize adenosine, and exogenous and cardiac fibroblast-derived adenosine inhibits cardiac fibroblast proliferation via activation of A2B receptors. Cardiac fibroblast-derived adenosine may regulate cardiac hypertrophy and/or remodeling by modulating cardiac fibroblast proliferation.

A1 adenosine receptors potently regulate heart rate in mammalian embryos.

A1 adenosine receptors (A1ARs) have been recently shown to be expressed in rodent embryonic hearts at very early stages of development. To determine the functional significance of fetal cardiac A1AR expression during embryogenesis, murine fetal heart preparations were studied between postconceptual days 9 and 12. Dose-response curves generated using a variety of adenosine agonists revealed that A1AR activation potently regulated fetal heart rates. The A1AR agonist, N6-cyclopentyladenosine, inhibited heart rates in a dose-dependent manner (half-maximal effective concentration = 3.6 x 10(-8) M) and stopped fetal cardiac contractions in 63% of preparations. In contrast, A2a and A2b receptor activation did not alter heart rates, and activation of A3 receptors produced modest declines in heart rates. Endogenous adenosine also acted tonically to suppress fetal heart rates, as demonstrated by the A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine, increasing heart rates, whereas the adenosine reuptake blocker dipyridamole lowered fetal heart rates. Pertussis toxin treatment blocked A1AR action, showing that A1AR action was G protein mediated. Using drugs that alter cAMP levels and ion channel action, we were able to show that A1AR action involves events mediated by cAMP, ATP-dependent K, L-type calcium, sodium, and chloride channels, and the pacemaker current. These data show that adenosine and A1ARs potently regulate mammalian heart rates via multiple effector systems at very early stages of prenatal development.