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A3 adenosine receptor signaling influences pulmonary inflammation and fibrosis.

ABSTRACT: Adenosine is a signaling molecule produced during conditions that cause cellular stress or damage. This signaling pathway is implicated in the regulation of pulmonary disorders through the selective engagement of adenosine receptors. The goal of this study was to examine the involvement of the A(3) adenosine receptor (A(3)R) in a bleomycin model of pulmonary inflammation and fibrosis. Results demonstrated that A(3)R-deficient mice exhibit enhanced pulmonary inflammation that included an increase in eosinophils. Accordingly, there was a selective up-regulation of eosinophil-related chemokines and cytokines in the lungs of A(3)R-deficient mice exposed to bleomycin. This increase in eosinophil numbers was accompanied by a decrease in the amount of extracellular eosinophil peroxidase activity in lavage fluid from A(3)R-deficient mice exposed to bleomycin, an observation suggesting that the A(3)R is necessary for eosinophil degranulation in this model. Despite an increase in inflammatory metrics associated with A(3)R-deficient mice treated with bleomycin, there was little difference in the degree of pulmonary fibrosis. Examination of fibrotic mediators demonstrated enhanced transforming growth factor (TGF)-beta1 expression, but not a concomitant increase in TGF-beta1 activity. This was associated with the loss of expression of matrix metalloprotease 9, an activator of TGF-beta1, in alveolar macrophages and airway mast cells in the lungs of A(3)R-deficient mice. Together, these results suggest that the A(3)R serves antiinflammatory functions in the bleomycin model, and is also involved in regulating the production of mediators that can impact fibrosis.

SUBMITTER: Morschl E

PROVIDER: S-EPMC2586045 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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A3 adenosine receptor signaling influences pulmonary inflammation and fibrosis.

Morschl Eva E Molina Jose G JG Volmer Jonathan B JB Mohsenin Amir A Pero Ralph S RS Hong Jeong-Soo JS Kheradmand Farrah F Lee James J JJ Blackburn Michael R MR

American journal of respiratory cell and molecular biology 20080627 6

Adenosine is a signaling molecule produced during conditions that cause cellular stress or damage. This signaling pathway is implicated in the regulation of pulmonary disorders through the selective engagement of adenosine receptors. The goal of this study was to examine the involvement of the A(3) adenosine receptor (A(3)R) in a bleomycin model of pulmonary inflammation and fibrosis. Results demonstrated that A(3)R-deficient mice exhibit enhanced pulmonary inflammation that included an increase ...[more]

PMID: 18587054

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Project description:Increasing evidence suggests that G protein-coupled receptors form functional dimers or larger oligomeric complexes through homo- or heterodimerization, and that various transmembrane (TM) domains contribute dimerization interfaces. In this study, monomeric receptor structures - either the monomeric crystallographic structure of bovine rhodopsin or an A(3) adenosine receptor (AR) homology model - were dimerized by computational methods assuming various TM contact regions, optimized, and compared. The semi-empirical oligomeric structure of mouse rhodopsin studied in a native disc membrane with atomic force microscopy was used to establish the distance between monomers in the initial dimeric models. Among eight variations of symmetrical homodimers of bovine rhodopsin, the favored dimeric assembly closely resembled the semi-empirical model, in which TM domains 4 and 5 were the contact site, thus validating this approach. We used similar methods to generate eight homodimers of the A(3)AR and found the favored dimeric interface similarly to be TM4-5. By this method, dimeric variations - TM1-2, TM2-3, TM2-4, TM3-4, TM4-5, TM5-6, TM6-7, and TM7-1 - were constructed with the SYBYL 7.0 program by using a novel "fit-centroids-normal" method. Fitting atoms considered one of eight TM-TM centroids or seven-TM centroids, two centroids of each monomer, and a normal atom passing through the plane containing all centroids. Following molecular dynamics, the most energetically favorable contact modes were identified. In addition to TM4-5, which was favored in both rhodopsin and A(3)AR dimeric models, TM1-2 dimers in which helices 8 also contacted each other were judged favorable. The largest contact surface area between the monomers among the various homodimers, determined by van der Waals calculation with the MOLCAD surface program, was for the TM4-5 dimer. This contact surface also showed a high degree of shape complementarity. In addition, the TM4-5 dimers made by this theoretical method were more stable than the semi-empirically determined dimer.

Project description:BACKGROUND:Adenosine is generated in response to cellular stress and damage and is elevated in the lungs of patients with chronic lung disease. Adenosine signaling through its cell surface receptors serves as an amplifier of chronic lung disorders, suggesting adenosine-based therapeutics may be beneficial in the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Previous studies in mouse models of chronic lung disease demonstrate that the key components of adenosine metabolism and signaling are altered. Changes include an up-regulation of CD73, the major enzyme of adenosine production and down-regulation of adenosine deaminase (ADA), the major enzyme for adenosine metabolism. In addition, adenosine receptors are elevated. METHODOLOGY/PRINCIPAL FINDINGS:The focus of this study was to utilize tissues from patients with COPD or IPF to examine whether changes in purinergic metabolism and signaling occur in human disease. Results demonstrate that the levels of CD73 and A(2B)R are elevated in surgical lung biopsies from severe COPD and IPF patients. Immunolocalization assays revealed abundant expression of CD73 and the A(2B)R in alternatively activated macrophages in both COPD and IPF samples. In addition, mediators that are regulated by the A(2B)R, such as IL-6, IL-8 and osteopontin were elevated in these samples and activation of the A(2B)R on cells isolated from the airways of COPD and IPF patients was shown to directly induce the production of these mediators. CONCLUSIONS/SIGNIFICANCE:These findings suggest that components of adenosine metabolism and signaling are altered in a manner that promotes adenosine production and signaling in the lungs of patients with COPD and IPF, and provide proof of concept information that these disorders may benefit from adenosine-based therapeutics. Furthermore, this study provides the first evidence that A(2B)R signaling can promote the production of inflammatory and fibrotic mediators in patients with these disorders.

Project description:BACKGROUND: An approach to use multivalent dendrimer carriers for delivery of nucleoside signaling molecules to their cell surface G protein-coupled receptors (GPCRs) was recently introduced. RESULTS: A known adenosine receptor (AR) agonist was conjugated to polyamidoamine (PAMAM) dendrimer carriers for delivery of the intact covalent conjugate to on the cell surface. Depending on the linking moiety, multivalent conjugates of the N6-chain elongated functionalized congener ADAC (N6-[4-[[[4-[[[(2-aminoethyl)amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]-adenosine) achieved unanticipated high selectivity in binding to the cytoprotective human A3 AR, a class A GPCR. The key to this selectivity of > 100-fold in both radioreceptor binding (Ki app = 2.4 nM) and functional assays (EC50 = 1.6 nM in inhibition of adenylate cyclase) was maintaining a free amino group (secondary) in an amide-linked chain. Attachment of neutral amide-linked chains or thiourea-containing chains preserved the moderate affinity and efficacy at the A1 AR subtype, but there was no selectivity for the A3 AR. Since residual amino groups on dendrimers are associated with cytotoxicity, the unreacted terminal positions of this A3 AR-selective G2.5 dendrimer were present as carboxylate groups, which had the further benefit of increasing water-solubility. The A3 AR selective G2.5 dendrimer was also visualized binding the membrane of cells expressing the A3 receptor but did not bind cells that did not express the receptor. CONCLUSION: This is the first example showing that it is feasible to modulate and even enhance the pharmacological profile of a ligand of a GPCR based on conjugation to a nanocarrier and the precise structure of the linking group, which was designed to interact with distal extracellular regions of the 7 transmembrane-spanning receptor. This ligand tool can now be used in pharmacological models of tissue rescue from ischemia and to probe the existence of A3 AR dimers.

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A3 adenosine receptor signaling influences pulmonary inflammation and fibrosis.

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A3 adenosine receptor signaling influences pulmonary inflammation and fibrosis.

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