A3 Adenosine Receptor Activation Triggers Phosphorylation of Protein Kinase B and Protects Rat Basophilic Leukemia 2H3 Mast Cells from Apoptosis
- Departments of 1Cardiovascular Medicine (Z.G., J.L.) and 2Molecular Physiology and Biological Physics (Y.-J.D., J.L.), University of Virginia, Charlottesville, Virginia; and 3Laboratory of Neurochemistry, National Institute on Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland (B.-S.L.)
Abstract
Adenosine accumulates to high levels in inflamed or ischemic tissues and activates A3 adenosine receptors (ARs) on mast cells to trigger degranulation. Here we show that stimulation of rat basophilic leukemia (RBL)-2H3 mast-like cells with the A3 AR agonistsN6-(3-iodo)benzyl-5′-N-methylcarboxamidodoadenosine (IB-MECA; 10 nM) or inosine (10 μM) stimulates phosphorylation of protein kinase B (Akt). IB-MECA (1 μM) also causes a >50% reduction in apoptosis caused by exposure of RBL-2H3 cells to UV light. Akt phosphorylation is not stimulated by 100 nMN6-cyclopentyladenosine (A1-selective) or CGS21680 (A2A-selective) and is absent in cells pretreated with wortmannin or pertussis toxin. TheKI values of the AR antagonists BW-1433 and 8-sulfophenyltheophylline (8-SPT) were determined in radioligand binding assays for all four subtypes of rat ARs: BW-1433 (A1, 5.8 ± 1.0 nM; A2A, 240 ± 37; A2B, 30 ± 10; A3, 12,300 ± 3,700); 8-SPT (A1, 3.2 ± 1.2 μM; A2A, 57 ± 4; A2B, 2.2 ± 0.8; A3, >100). BW-1433 and the A3-slective antagonist MRS1523 (5 μM), but not 8-SPT (100 μM), block IB-MECA-induced protection from apoptosis, confirming the A3 AR as the mediator of the antiapoptotic response. The data suggest that adenosine and inosine activate Gi-coupled A3 ARs to protect mast cells from apoptosis by a pathway involving the βγ subunits of Gi, phosphatidylinositol 3-kinase β, and Akt. We speculate that activation of A3ARs on mast cells or other cells that express A3 ARs (e.g., eosinophils) may facilitate their survival and accumulation in inflamed tissues.
Footnotes
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Send reprint requests to: Joel Linden, Ph.D., Box MR4 6012 Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908-0466. E-mail: jlinden{at}virginia.edu
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↵1 Current address: CV Therapeutics, Palo Alto, California.
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This work was supported by Grant R01-HL37942 from the National Institutes of Health.
- Abbreviations:
- Akt or PKB
- protein kinase B
- PI3K
- phosphatidylinositol 3-kinases
- AR
- adenosine receptor
- RBL
- rat basophilic leukemia
- IB-MECA
- N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine
- CPA
- N6-cyclopentyladenosine
- CGS21680
- 2-p-(2-carboxyethyl)phenethylamino-5′-ethylcarboxaminoadenosine
- 8-SPT
- 8-sulfophenyltheophylline
- enprofylline
- 3-propylxanthine
- MRS1523
- 5-propyl 2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate
- WRC-0571
- C8-(N-methylisopropyl)-amino-N6-(5-endohydroxy)-endonorbornan-2-yl-9-methyladenine
- BW-1433
- 8-(4-carboxyethenylphenyl)-1,3-dipropylxanthine
- ABA
- N6-aminobenzyladenosine
- HEK
- human embryonic kidney
- HE
- HEPES/EDTA
- ZM241385
- 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol
- TUNEL
- terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein isothiocyanate nick end labeling
- DMSO
- dimethylsulfoxide
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- Received May 17, 2000.
- Accepted May 10, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
