Induction of Multiple Effects on Adenylyl Cyclase Regulation by Chronic Activation of the Human A3 Adenosine Receptor
- Departments of 1Medicine and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710 (T.M.P., G.L.S.), and 2Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage, Hertfordshire SG1 2NY, UK (C.A.H., J.C.)
Abstract
The A3 adenosine receptor (A3AR) contributes to several cardiovascular effects of adenosine, including antihypertensive and cardioprotective effects. Although several studies have detailed the mechanisms underlying agonist-mediated desensitization of the rat A3AR, the regulation of the human A3AR, which displays only a 70% amino acid identity with the rat homologue, has not been addressed. Using a Chinese hamster ovary cell line stably expressing a recombinant human A3AR, we demonstrated that prolonged treatment with the AR agonist 5′-N-ethylcarboxamidoadenosine induces uncoupling of the A3AR from G proteins and functional desensitization. In addition to A3AR desensitization, a 1.5–2.5-fold increase was noted in the adenylyl cyclase (AC) activity achieved in the presence of GTP with or without forskolin. This sensitization of AC activity was not a consequence of the down-regulation of Giproteins induced by NECA treatment and was not associated with sustained or transient increases in the expression of Gs. Time course experiments revealed that the onset of sensitization was half-maximal between 2 and 3 hr but was not due to the synthesis of new proteins because cycloheximide treatment failed to inhibit sensitization. The inability of the sensitization process to alter the AC activity obtained in the presence of manganese chloride suggests that prolonged A3AR activation increases the coupling efficiency between Gs and AC catalytic units. This phenomenon has implications for long term cellular adaptation to agonist because in agonist-treated cells, the extent to which a suboptimal concentration of forskolin could increase phosphorylation of the cAMP-responsive element binding protein was elevated compared with vehicle-treated controls.
Footnotes
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Send reprint requests to: Timothy M. Palmer, Ph.D., Duke University Medical Center, Box 3444 (Dept. of Medicine), Durham, NC 27710. E-mail: tmp3c{at}udcf.gla.ac.uk
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↵1 Current affiliation: Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, UK
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↵2 T. M. Palmer, unpublished observations.
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This work was supported by National Heart, Lung, and Blood Institute Specialized Center of Organized Research Grant 5-P50-HL54314 in Ischemic Disease (G.L.S.), by the American Heart Association, North Carolina Affiliate (T.M.P.), and by Glaxo Wellcome (T.M.P.).
- Abbreviations:
- AR
- adenosine receptor
- CHO
- Chinese hamster ovary
- AB-MECA
- N6-(4-aminobenzyl)-5′-N-methylcarboxamidoadenosine
- IB-MECA
- N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- PAGE
- polyacrylamide gel electrophoresis
- CREB
- cAMP-responsive element-binding protein
- NECA
- 5′-N-ethylcarboxamidoadenosine
- AC
- adenylyl cyclase
- PTX
- pertussis toxin
- PKC
- protein kinase C
- SDS
- sodium dodecyl sulfate
- ATF-1
- activating transcription factor-1
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- Received May 13, 1997.
- Accepted July 8, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
