The Vascular Smooth Muscle Type I Angiotensin II Receptor mRNA Is Destabilized by Cyclic AMP-Elevating Agents
Abstract
Although processes involved in mRNA degradation play a significant role in dictating steady state mRNA levels, the influence of cell surface signaling on mRNA stability control is understood incompletely. In this study, the effects of cAMP-elevating agents on type I angiotensin II receptor (AT1-R) mRNA levels were assessed in cultured rat aortic vascular smooth muscle cells (VSMCs). AT1-R mRNA levels are rapidly reduced by forskolin treatment, in which the maximal effect yields an 80% reduction in AT1-R mRNA levels after 6 hr of treatment. The rate of AT1-R mRNA decay in response to forskolin is greater than its apparent intrinsic decay, as assessed in the presence of the transcriptional inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole, suggesting forskolin treatment destabilizes the AT1-R mRNA. Nuclear run-on analysis indicates forskolin treatment does not affect transcription of the AT1-R gene in VSMCs, implying induced AT1-R mRNA destabilization accounts for the entire effect of forskolin in decreasing AT1-R mRNA levels. Dose-effect studies that assessed AT1-R mRNA levels and cAMP production were conducted using forskolin and the β-adrenergic receptor agonist isoproterenol as agonists. Isoproterenol is almost 3 orders of magnitude more potent at eliciting the reduction in AT1-receptor mRNA levels than it is at stimulating cAMP production. Similarly, forskolin elicits reductions in AT1-R mRNA, which occur at concentrations that fail to elicit a detectable production of cAMP. However, protein kinase A activity is stimulated maximally by isoproterenol and forskolin concentrations that do not stimulate detectable cAMP production. These data provide evidence that the mechanism for down-regulation of AT1-R mRNA levels by cAMP-elevating agents in VSMCs occurs via a PKA-regulated mRNA destabilization pathway.
Footnotes
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Send reprint requests to: T. J. Murphy, Ph.D., Department of Pharmacology, Emory University School of Medicine, 5031 O.W. Rollins Research Building, Atlanta, GA 30322. E-mail:medtjm{at}bimcore.emory.edu
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↵1 X. Wang, G. Nickenig, and T. J. Murphy, unpublished observations.
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↵2 X. Wang and T. J. Murphy, data not shown.
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This work was supported in part by National Institutes of Health Grants HL48252 and HL52810 and by Grants-in-Aid from the American Heart Association and Sanofi-Winthrop. G.N. is supported by Deutsche Forschungsgemeinschaft Grant DFG Ni398/1–1. T.J.M. is an Established Investigator of the American Heart Association.
- Abbreviations:
- VSMC
- vascular smooth muscle cell
- DRB
- 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole
- Bt2
- dibutyryl
- AT1-R
- type I angiotensin II receptor
- PKA
- protein kinase A
- DMSO
- dimethylsulfoxide
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- SSC
- standard saline citrate
- SDS
- sodium dodecyl sulfate
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
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- Received April 1, 1997.
- Accepted July 21, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
