Inhibition of Cyclic AMP-Dependent Kinase by Expression of a Protein Kinase Inhibitor/Enhanced Green Fluorescent Fusion Protein Attenuates Angiotensin II-Induced Type 1 AT1 Receptor mRNA Down-Regulation in Vascular Smooth Muscle Cells
- Department of Pharmacology, Emory University School of Medicine, and Program in Molecular Therapeutics and Toxicology, Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, Georgia 30322
Abstract
Expression of the angiotensin II type 1 receptor (AT1-R) mRNA in vascular smooth muscle cells (VSMC) is down-regulated by a variety of agonists, including growth factors, agonists of Gαq protein-coupled receptors, and activators of adenylyl cyclase. To determine whether cAMP-dependent protein kinases (PKA) participates in AT1-R mRNA down-regulation controlled by multiple classes of receptors, a PKA inhibitor peptide (PKIα) was developed and expressed in rat VSMC as a fusion with the enhanced green fluorescent protein (eGFP). PKA activity elicited both by forskolin and angiotensin II is suppressed in cells expressing this fusion protein (PKIα-eGFP), but platelet-derived growth factor-BB does not stimulate PKA activity in this preparation. PKIα-eGFP expression fully inhibits the forskolin-stimulated down-regulation of AT1-R mRNA levels and blocks 50% of the effect elicited by angiotensin II. This indicates that PKA plays a substantial role in angiotensin II-stimulated AT1-R mRNA down-regulation. However, inhibition of PKA has no effect on AT1-R mRNA down-regulation caused by platelet-derived growth factor-BB. These findings show how agonists such as angiotensin II that are not normally considered as activators of PKA can use PKA-dependent processes to modulate gene expression. These findings also provide definitive evidence that PKA-dependent pathways are involved in modulation of AT1-R mRNA levels in VSMC.
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:tmurphy{at}pharm.emory.edu
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This work is supported by National Institutes of Health Grants HL52810, HL56107, and NS32706. T.J.M. is an Established Investigator of the American Heart Association.
- Abbreviations:
- VSMC
- vascular smooth muscle cells
- AT1-R
- type-1 angiotensin II receptor
- PKIα
- heat stabile cAMP-dependent protein kinase inhibitor
- PKA
- cAMP-dependent protein kinase
- eGFP
- enhanced green fluorescent protein
- tTA
- tetracycline transactivator
- LTR
- long terminal repeat
- PKG
- cGMP-dependent protein kinase
- DMEM
- Dulbecco’s modified Eagle’s medium
- FACScan
- fluorescent activated cell scan analysis
- FACS
- fluorescent activated cell sorting
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- PDGF-BB
- platelet-derived growth factor, BB isoform
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- Received March 23, 1998.
- Accepted May 26, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
