Other search tools

About this data

The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.

If you have any questions or comments, please contact us.

Results: 1 to 10 of 18

Publication Record

Connections

Development of a reliable automated screening system to identify small molecules and biologics that promote human β-cell regeneration.
Aamodt KI, Aramandla R, Brown JJ, Fiaschi-Taesch N, Wang P, Stewart AF, Brissova M, Powers AC
(2016) Am J Physiol Endocrinol Metab 311: E859-E868
MeSH Terms: Activins, Adenosine, Adenosine A2 Receptor Agonists, Adenosine-5'-(N-ethylcarboxamide), Adult, Automation, Cell Culture Techniques, Cell Proliferation, Drug Evaluation, Preclinical, Erythropoietin, Exenatide, Female, GABA Agents, Harmine, Humans, Incretins, Insulin-Secreting Cells, Male, Middle Aged, Monoamine Oxidase Inhibitors, Myostatin, Nucleosides, Peptides, Platelet-Derived Growth Factor, Prolactin, Regeneration, Serotonin, Serotonin Receptor Agonists, Vasodilator Agents, Venoms, Young Adult, gamma-Aminobutyric Acid
Show Abstract · Added April 26, 2017
Numerous compounds stimulate rodent β-cell proliferation; however, translating these findings to human β-cells remains a challenge. To examine human β-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human β-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled β-cells with high specificity using both nuclear and cytoplasmic markers. β-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1β signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67 β-cells, whereas treatment with other compounds had limited to no effect on human β-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human β-cell proliferation, thus allowing for increased testing of candidate human β-cell mitogens.
0 Communities
2 Members
0 Resources
32 MeSH Terms
Rapid stimulation of presynaptic serotonin transport by A(3) adenosine receptors.
Zhu CB, Steiner JA, Munn JL, Daws LC, Hewlett WA, Blakely RD
(2007) J Pharmacol Exp Ther 322: 332-40
MeSH Terms: Adenosine, Adenosine-5'-(N-ethylcarboxamide), Animals, Biological Transport, Cyclic GMP-Dependent Protein Kinases, MAP Kinase Signaling System, Male, Mice, Mice, Inbred C57BL, Rats, Rats, Sprague-Dawley, Receptor, Adenosine A3, Serotonin, Serotonin Plasma Membrane Transport Proteins, Synaptosomes, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added July 10, 2013
The inactivation of synaptic serotonin (5-hydroxytryptamine, 5-HT) is largely established through the actions of the presynaptic, antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). Recent studies have demonstrated post-translational regulation of SERT mediated by multiple Ser/Thr kinases, including protein kinases C and G (PKC and PKG) and p38 mitogen-activated protein kinase (MAPK), as well as the Ser/Thr phosphatase PP2A. Less well studied are specific surface receptors that target these signaling pathways to control SERT surface expression and/or catalytic rates. Using rat basophilic leukemia 2H3 cell line (RBL-2H3), we previously established that activation of A(3) adenosine receptors (A(3)AR) stimulates SERT activity via both PKG and p38 MAPK (Zhu et al., 2004a). Whether A(3)ARs regulate SERT in the central nervous system (CNS) is unknown. Here we report that the A(3)AR agonist N(6)-(3-iodobenzyl)-N-methyl-5'carbamoyladenosine (IB-MECA) rapidly (10 min) and selectively stimulates 5-HT transport in mouse midbrain, hippocampal, and cortical synaptosomes. IB-MECA-induced stimulation of 5-HT uptake is blocked by the selective A(3)AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(+/-)dihydropyridine-3,5-dicarboxylate (MRS1191) and is absent from synaptosomes prepared from A(3)AR knockout mice. Kinetic analyses demonstrate that IB-MECA induces an increase of 5-HT transport V(max) with no significant change in K(m). As in RBL-2H3 cells, IB-MECA stimulation of synaptosomal 5-HT uptake can be blocked by preincubation with PKG antagonists N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) and DT-2 (YGRKKRRQRRRPPLRK(5)H), as well as by the p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Chronoamperometry studies in the anesthetized rat hippocampus support a role for A(3)ARs in SERT regulation in vivo. Together, these results identify a novel, region-specific action of CNS A(3)ARs in the modulation of SERT-mediated 5-HT transport that may be relevant for the etiology and/or therapy of 5-HT-linked brain disorders.
1 Communities
1 Members
0 Resources
16 MeSH Terms
Role of adenosine receptors in the regulation of angiogenic factors and neovascularization in hypoxia.
Ryzhov S, McCaleb JL, Goldstein AE, Biaggioni I, Feoktistov I
(2007) J Pharmacol Exp Ther 320: 565-72
MeSH Terms: Adenosine, Adenosine-5'-(N-ethylcarboxamide), Animals, Cells, Cultured, Gene Expression Regulation, Hindlimb, Humans, Hypoxia, Interleukin-8, Ischemia, Male, Mice, Mice, Inbred C57BL, Neovascularization, Physiologic, RNA, Messenger, Receptors, Purinergic P1, Vascular Endothelial Growth Factor A
Show Abstract · Added December 10, 2013
Because hypoxia increases extracellular adenosine levels and stimulates angiogenesis, we evaluated the relative roles of reduced oxygen concentrations and adenosine receptor activation in the production of angiogenic factors. In vitro, we analyzed the effects of hypoxia and adenosine on the secretion of angiogenic factors from human microvascular endothelial cells (HMEC-1). To study the effects of hypoxia alone, we scavenged adenosine from the hypoxic medium with adenosine deaminase, and we used the stable adenosine analog 5'-N-ethylcarboxamidoadenosine (NECA) to study the effects of stimulation of adenosine receptors. In the absence of adenosine, hypoxia stimulated vascular endothelial growth factor (VEGF) but not interleukin-8 (IL-8) secretion from HMEC-1. In contrast, NECA stimulated both VEGF and IL-8 secretion. VEGF secretion was increased 1.9 +/- 0.04-fold with NECA (10 microM) and 1.7 +/- 0.1-fold with hypoxia (5% O(2)) but 3.8 +/- 0.1-fold when these two stimuli were combined. Thus, adenosine receptors act in a cooperative fashion with hypoxia to stimulate VEGF and induce IL-8 secretion not stimulated by hypoxia alone. In vivo, antagonism of adenosine receptors with caffeine abrogated VEGF up-regulation induced by local injection of NECA into the mouse hind limb and produced a 46% reduction of neovascularization in a mouse ischemic hind limb model. Our study suggests that adenosine actions are not redundant but rather are complementary to the direct effects of hypoxia. Stimulation of adenosine receptors not only contributes to the overall effect of hypoxia but also has additional actions in the regulation of angiogenic factors. Thus, adenosine receptors represent a potential therapeutic target for regulation of neovascularization.
0 Communities
2 Members
0 Resources
17 MeSH Terms
Cross-talk between G(s)- and G(q)-coupled pathways in regulation of interleukin-4 by A(2B) adenosine receptors in human mast cells.
Ryzhov S, Goldstein AE, Biaggioni I, Feoktistov I
(2006) Mol Pharmacol 70: 727-35
MeSH Terms: Adenosine-5'-(N-ethylcarboxamide), Cells, Cultured, Colforsin, GTP-Binding Protein alpha Subunits, Gq-G11, GTP-Binding Protein alpha Subunits, Gs, Humans, Interleukin-4, Mast Cells, NFATC Transcription Factors, Receptor, Adenosine A2B, Signal Transduction, Up-Regulation
Show Abstract · Added December 10, 2013
Human mast cells express functional A(2A) and A(2B) adenosine receptors. However, only stimulation of A(2B), not A(2A), leads to secretion of interleukin (IL)-4, an important step in adenosine receptor-mediated induction of IgE synthesis by B-cells. In this study, we investigate intracellular pathways that link stimulation of A(2B) receptors to IL-4 up-regulation in HMC-1 mast cells. Both A(2A) and A(2B) receptors couple to G(s) proteins and stimulate adenylate cyclase, but only A(2B) stimulates phospholipase Cbeta through coupling to G(q) proteins leading to activation of protein kinase C and calcium mobilization. Inhibition of phospholipase Cbeta completely blocked A(2B) receptor-dependent IL-4 secretion. The protein kinase C inhibitor 2-{8-[(dimethylamino)-methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide (Ro-32-0432) had no effect on A(2B) receptor-mediated IL-4 secretion but inhibited phorbol 12-myristate 13-acetate-stimulated IL-4 secretion. In contrast, chelation of intracellular Ca(2+) inhibited both A(2B) receptor- and ionomycin-dependent IL-4 secretion. This Ca(2+)-sensitive pathway probably includes calcineurin and nuclear factor of activated T cells, because A(2B) receptor-dependent IL-4 secretion was blocked with cyclosporin A or 11R-VIVIT peptide. G(s)-linked pathways also play a role in the A(2B) receptor-dependent stimulation of IL-4 secretion; inhibition of adenylate cyclase or protein kinase A attenuated A(2B) receptor-dependent IL-4 secretion. Although stimulation of adenylate cyclase with forskolin did not increase IL-4 secretion on its own, it potentiated the effect of Pasteurella multocida toxin by 2-fold and ionomycin by 3-fold. Both forskolin and stimulation of A(2B) receptors up-regulated NFATc1 protein levels. We conclude that A(2B) receptors up-regulate IL-4 through G(q) signaling that is potentiated via cross-talk with G(s)-coupled pathways.
0 Communities
2 Members
0 Resources
12 MeSH Terms
Adenosine-activated mast cells induce IgE synthesis by B lymphocytes: an A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma.
Ryzhov S, Goldstein AE, Matafonov A, Zeng D, Biaggioni I, Feoktistov I
(2004) J Immunol 172: 7726-33
MeSH Terms: Adenosine, Adenosine-5'-(N-ethylcarboxamide), Asthma, B-Lymphocytes, Cell Communication, Cells, Cultured, Coculture Techniques, Cytokines, Humans, Immunoglobulin E, Interleukin-13, Interleukin-4, Mast Cells, RNA, Messenger, Receptor, Adenosine A2B, Th2 Cells, Up-Regulation
Show Abstract · Added December 10, 2013
Adenosine provokes bronchoconstriction in asthmatics through acute activation of mast cells, but its potential role in chronic inflammation has not been adequately characterized. We hypothesized that adenosine up-regulates Th2 cytokines in mast cells, thus promoting IgE synthesis by B lymphocytes. We tested this hypothesis in human mast cells (HMC-1) expressing A(2A), A(2B), and A(3) adenosine receptors. The adenosine analog 5'-N-ethylcarboxamidoadenosine (NECA) (10 microM) increased mRNA expression of IL-1beta, IL-3, IL-4, IL-8, and IL-13, but not IL-2 and IFN-gamma. Up-regulation of IL-4 and IL-13 was verified using RT-PCR and ELISA; 10 microM NECA increased IL-13 concentrations in HMC-1 conditioned medium 28-fold, from 7.6 +/- 0.3 to 215 +/- 4 pg/ml, and increased IL-4 concentrations 6-fold, from 19.2 +/- 0.1 to 117 +/- 2 pg/ml. This effect was mediated by A(2B) receptors because neither the selective A(2A) agonist 2-p-(2-carboxyethyl)phenethylamino-NECA nor the selective A(3) agonist N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine reproduced it, and the selective A(2B) antagonist 3-isobutyl-8-pyrrolidinoxanthine prevented it. Constitutive expression of CD40 ligand on HMC-1 surface was not altered by NECA. Human B lymphocytes cocultured for 12 days with NECA-stimulated HMC-1 produced 870 +/- 33 pg IgE per 10(6) B cells, whereas lymphocytes cocultured with nonstimulated HMC-1, or cultured alone in the absence or in the presence of NECA, produced no IgE. Thus, we demonstrated induction of IgE synthesis by the interaction between adenosine-stimulated mast cells and B lymphocytes, and suggest that this mechanism is involved in the amplification of the allergic inflammatory responses associated with asthma.
0 Communities
2 Members
0 Resources
17 MeSH Terms
Adenosine receptor, protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation of serotonin transporters involves both transporter trafficking and activation.
Zhu CB, Hewlett WA, Feoktistov I, Biaggioni I, Blakely RD
(2004) Mol Pharmacol 65: 1462-74
MeSH Terms: Adenosine-5'-(N-ethylcarboxamide), Animals, Biological Transport, CHO Cells, Calcium, Carrier Proteins, Cells, Cultured, Cricetinae, Cyclic GMP, Cyclic GMP-Dependent Protein Kinases, Female, Guanylate Cyclase, Membrane Glycoproteins, Membrane Transport Proteins, Mitogen-Activated Protein Kinases, Nerve Tissue Proteins, Phosphodiesterase Inhibitors, Piperazines, Purines, Rats, Receptors, Purinergic P1, Serotonin, Serotonin Plasma Membrane Transport Proteins, Sildenafil Citrate, Sulfones, Type C Phospholipases, Up-Regulation, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added December 10, 2013
Serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) are critical determinants of synaptic 5-HT inactivation and the targets for multiple drugs used to treat psychiatric disorders. In support of prior studies, we found that short-term (5-30 min) application of the adenosine receptor (AR) agonist 5'-N-ethylcarboxamidoadenosine (NECA) induces an increase in 5-HT uptake Vmax in rat basophilic leukemia 2H3 cells that is enhanced by pretreatment with the cGMP phosphodiesterase inhibitor sildenafil. NECA stimulation is blocked by the A3 AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(+/-)dihydropyridine-3,5-dicarboxylate (MRS1191), by the phospholipase C inhibitor 1-(6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl)-1H-pyrrole-2,5-dione (U73122), by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, and by the guanyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Hydroxylamine, a nitric-oxide donor, and 8-bromo-cGMP, a membrane-permeant analog of cGMP, mimic the effects of NECA on 5-HT uptake, whereas the protein kinase G (PKG) inhibitor N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) blocks NECA, hydroxylamine, and 8-bromo-cGMP effects. NECA stimulation activates p38 mitogen-activated protein kinase (MAPK), whereas p38 MAPK inhibitors block NECA stimulation of SERT activity, as does the protein phosphatase 2A (PP2A) inhibitor calyculin A. 5-HT-displaceable [125I]3beta-(4-iodophenyl)-tropane-2beta-carboxylic acid methylester tartrate (RTI-55) whole-cell binding is increased by NECA or sildenafil, and both surface binding and cell surface SERT protein are elevated after NECA or sildenafil stimulation of AR/SERT-cotransfected Chinese hamster ovary cells. Whereas p38 MAPK inhibition blocks NECA stimulation of 5-HT activity, it fails to blunt stimulation of SERT surface density. Moreover, inactivation of existing surface SERTs fails to eliminate NECA stimulation of SERT. Together, these results reveal two PKG-dependent pathways supporting rapid SERT regulation by A3 ARs, one leading to enhanced SERT surface trafficking, and a separate, p38 MAPK-dependent process augmenting SERT intrinsic activity.
1 Communities
3 Members
0 Resources
28 MeSH Terms
Inhibition of human mast cell activation with the novel selective adenosine A(2B) receptor antagonist 3-isobutyl-8-pyrrolidinoxanthine (IPDX)(2).
Feoktistov I, Garland EM, Goldstein AE, Zeng D, Belardinelli L, Wells JN, Biaggioni I
(2001) Biochem Pharmacol 62: 1163-73
MeSH Terms: Adenosine, Adenosine-5'-(N-ethylcarboxamide), Anti-Asthmatic Agents, Cells, Cultured, Humans, Mast Cells, Purinergic P1 Receptor Antagonists, Pyrrolidinones, Receptor, Adenosine A2B, Receptors, Purinergic P1, Structure-Activity Relationship, Vasodilator Agents, Xanthine, Xanthines
Show Abstract · Added December 10, 2013
The antiasthmatic drug enprofylline was the first known selective, though not potent, A(2B) antagonist. On the basis of structure-activity relationships (SARs) of xanthine derivatives, we designed a novel selective adenosine A(2B) receptor antagonist, 3-isobutyl-8-pyrrolidinoxanthine (IPDX), with potency greater than that of enprofylline. IPDX displaced [3H]ZM241385 ([3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a]-[1,3,5]triazin-5-ylamino]ethyl)phenol) from human A(2B) adenosine receptors with a K(i) value of 470 +/- 2 nM and inhibited A(2B)-dependent cyclic AMP (cAMP) accumulation in human erythroleukemia (HEL) cells with a K(B) value of 625 +/- 71 nM. We found that IPDX was more selective than enprofylline toward human A(2B) receptors. It was 38-, 55-, and 82-fold more selective for human A(2B) than for human A(1) (K(i) value of 24 +/- 8 microM), human A(2A) (K(B) value of 36 +/- 8 microM), and human A(3) (K(i) value of 53 +/- 10 microM) adenosine receptors, respectively. IPDX inhibited NECA (5'-N-ethylcarboxamidoadenosine)-induced interleukin-8 secretion in human mast cells (HMC-1) with a potency close to that determined for A(2B)-mediated cAMP accumulation in HEL cells, thus confirming the role of A(2B) adenosine receptors in mediating human mast cell activation. Since adenosine triggers bronchoconstriction in asthmatic patients through human mast cell activation, IPDX may become a basis for the development of new antiasthmatic drugs with improved properties compared with those of enprofylline. Our data demonstrate that IPDX can be used as a tool to differentiate between A(2B) and other adenosine receptor-mediated responses.
0 Communities
2 Members
0 Resources
14 MeSH Terms
Proliferation, migration, and ERK activation in human retinal endothelial cells through A(2B) adenosine receptor stimulation.
Grant MB, Davis MI, Caballero S, Feoktistov I, Biaggioni I, Belardinelli L
(2001) Invest Ophthalmol Vis Sci 42: 2068-73
MeSH Terms: Adenosine-5'-(N-ethylcarboxamide), Blotting, Western, Capillaries, Cell Count, Cell Division, Cell Movement, Cells, Cultured, Cyclic AMP Response Element-Binding Protein, Dose-Response Relationship, Drug, Endothelium, Vascular, Enzyme Activation, Enzyme Inhibitors, Humans, Mitogen-Activated Protein Kinases, Purinergic P1 Receptor Antagonists, Receptor, Adenosine A2B, Receptors, Purinergic P1, Retinal Vessels, Signal Transduction, Vasodilator Agents
Show Abstract · Added December 10, 2013
PURPOSE - The nucleoside adenosine has been implicated in angiogenesis. A previous study demonstrated that activation of the A(2B) adenosine receptor (AdoR) increases cAMP accumulation, cell proliferation, and VEGF expression in human retinal endothelial cells (HRECs). In the present study, the role of this receptor was further characterized by examination of the effects of the selective A(2B) AdoR antagonists 3-N-propylxanthine (enprofylline) and 3-isobutyl-8-pyrrolidinoxanthine (IPDX) on AdoR-mediated HREC proliferation, capillary tube formation, and signal-transduction pathways.
METHODS - HRECs were exposed to the adenosine analogue 5'-N-ethylcarboxamido-adenosine (NECA) in the absence or presence of AdoR antagonists. Migration was measured using Boyden chambers. Proliferation was assessed by counting cells. Western analysis was used to assess extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) in cell lysates. The effect of AdoR activation on tube formation was studied using cells grown on a synthetic basement membrane matrix.
RESULTS - NECA induced proliferation in a concentration-dependent manner that was inhibited by enprofylline and IPDX. NECA stimulated chemotaxis in a concentration-dependent manner that was also blocked by both A(2B) AdoR antagonists. NECA activated ERK and CREB in HRECs. Both A(2B) AdoR antagonists diminished activation of ERK by NECA exposure. ERK activation was also blocked by the ERK-mitogen-activated protein kinase (MAPK) inhibitor PD98059, but not by the protein kinase A (PKA) inhibitor H-89. CREB activation was blocked by H-89, but not by PD98059, suggesting that ERK activation is independent of PKA. NECA enhanced tube formation on the matrix, whereas both A(2B) AdoR antagonists attenuated this effect.
CONCLUSIONS - The selective A(2B) AdoR antagonists, enprofylline and IPDX, inhibited NECA-stimulated proliferation, ERK activation, cell migration, and capillary tube formation. A(2B) AdoR inhibition may offer a way to inhibit retinal angiogenesis and provide a novel therapeutic approach to treatment of diseases associated with aberrant neovascularization, such as diabetic retinopathy and retinopathy of prematurity.
0 Communities
2 Members
0 Resources
20 MeSH Terms
Adenosine A(2A) and A(2B) receptors in cultured human and porcine coronary artery endothelial cells.
Olanrewaju HA, Qin W, Feoktistov I, Scemama JL, Mustafa SJ
(2000) Am J Physiol Heart Circ Physiol 279: H650-6
MeSH Terms: 2-Chloroadenosine, Adenosine, Adenosine-5'-(N-ethylcarboxamide), Affinity Labels, Animals, Binding, Competitive, Cells, Cultured, Coronary Vessels, Cyclic AMP, Endothelium, Vascular, Gene Expression Regulation, Humans, Iodine Radioisotopes, Iodobenzenes, Kinetics, Phenethylamines, Purinergic P1 Receptor Antagonists, Radioligand Assay, Receptor, Adenosine A2A, Receptor, Adenosine A2B, Receptors, Purinergic P1, Reverse Transcriptase Polymerase Chain Reaction, Swine, Transcription, Genetic, Triazines, Triazoles
Show Abstract · Added May 29, 2014
We investigated the role of the cAMP link to the signal transduction mechanism coupled with adenosine A(2A) and A(2B) receptors in cultured human coronary artery endothelial cells (HCAEC) and porcine coronary artery endothelial cells (PCAEC). 2-[4-[2-¿2-[(4-aminophenyl)methylcarbonylamino]ethylaminocarbon yl¿eth yl]phenyl]ethylamino-5'- ethylcarboxamidoadenosine ((125)I-PAPA-APEC) (PAPA-APEC) was used to demonstrate the specific binding in PCAEC membranes. The specific binding was saturable and reversible with a maximal number of binding sites (B(max)) of 240 fmol/mg protein, and scatchard analysis revealed a single class of binding site with an equilibrium dissociation constant (K(d)) of 1. 17 +/- 0.035 nM. In competition experiments, adenosine receptor agonists showed the following order of potency (based on IC(50)): 5'-(N-ethylcarboxamido)adenosine (NECA) >/= CGS-21680 > 2-chloroadenosine. This order appears to be consistent with the A(2) adenosine receptor classification. We also studied the effects of adenosine agonists on the accumulation of cAMP as an indirect approach to show the presence of functional A(2) receptors. Similarly, the same adenosine agonists (10(-7)-10(-4) M) elicited the production of cAMP in intact endothelial cells in a dose-dependent manner, exhibiting consistently with the A(2) adenosine receptor classification. A selective A(2A) adenosine receptor antagonist (ZM-241385, 10(-8) M) significantly inhibited the effect of CGS-21680 on cAMP but only partly inhibited the effect of NECA, suggesting the presence of both A(2A) and A(2B) receptors. Western blot analysis further showed the immunoreactivity of A(2A) and A(2B) receptor at 45 and 36 kDa, respectively, in both HCAEC and PCAEC. Direct evidence for the presence of A(2A) and A(2B) receptors in cultured HCAEC and PCAEC by reverse transcription-polymerase chain reaction (RT-PCR), revealed expected PCR product sizes (205 and 173 bp) for A(2A) and A(2B) receptors in HCAEC and PCAEC, respectively. The data show that adenylate cyclase-coupled adenosine A(2A) and A(2B) receptors are present in coronary endothelial cells.
0 Communities
1 Members
0 Resources
26 MeSH Terms
A galpha(s) carboxyl-terminal peptide prevents G(s) activation by the A(2A) adenosine receptor.
Mazzoni MR, Taddei S, Giusti L, Rovero P, Galoppini C, D'Ursi A, Albrizio S, Triolo A, Novellino E, Greco G, Lucacchini A, Hamm HE
(2000) Mol Pharmacol 58: 226-36
MeSH Terms: Adenosine, Adenosine-5'-(N-ethylcarboxamide), Animals, Binding, Competitive, Cell Membrane, GTP-Binding Protein alpha Subunits, Gs, In Vitro Techniques, Magnetic Resonance Spectroscopy, Male, Molecular Sequence Data, Peptides, Phenethylamines, Protein Conformation, Pyrimidines, Radioligand Assay, Rats, Rats, Sprague-Dawley, Receptor, Adenosine A2A, Receptors, Purinergic P1, Signal Transduction, Triazoles, Tritium
Show Abstract · Added December 10, 2013
The molecular mechanisms of interaction between G(s) and the A(2A) adenosine receptor were investigated using synthetic peptides corresponding to various segments of the Galpha(s) carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [(3)H]2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxam idoade nosine ([(3)H]CGS21680), to A(2A) adenosine receptors in rat striatal membranes. The Galpha(s) peptides stimulated specific binding both in the presence and absence of 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS). Three peptides, Galpha(s)(378-394)C(379)A, Galpha(s)(376-394)C(379)A, and Galpha(s)(374-394)C(379)A, were the most effective. In the presence of GTPgammaS, peptide Galpha(s)(374-394)C(379)A increased specific binding in a dose-dependent fashion. However, the peptide did not stabilize the high-affinity state of the A(2A) adenosine receptor for [(3)H]CGS21680. Binding assays with a radiolabeled selective antagonist, [(3)H]5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH58261), showed that the addition of the Galpha(s) peptide modified the slope of the 5'-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTPgammaS, the displacement curve was right-shifted, whereas the addition of Galpha(s)(374-394)C(379)A caused a partial left-shift. Both curves were fitted by one-site models. This same Galpha(s) peptide was also able to disrupt G(s)-coupled signal transduction as indicated by inhibition of the A(2A) receptor-stimulated adenylyl cyclase activity without affecting either basal or forskolin-stimulated enzymatic activity in the same membrane preparations. Shorter peptides from Galpha(s) and Galpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide Galpha(s)(374-394)C(379)A to assume a compact carboxyl-terminal alpha-helical conformation in solution. Overall, our results point out the conformation requirement of Galpha(s) carboxyl-terminal peptides to modulate agonist binding to rat A(2A) adenosine receptors and disrupt signal transduction.
0 Communities
1 Members
0 Resources
22 MeSH Terms