α1-Adrenergic Receptor Activation of c-fos Expression in Transfected Rat-1 Fibroblasts: Role of Ca2+ 1
- Veterans Affairs Palo Alto Health Care System, Geriatrics Research, Education and Clinical Center, Palo Alto, California; and Department of Medicine, Stanford University, Stanford, California
Abstract
α1-Adrenergic receptors mediate mitogenic responses and increase intracellular free Ca2+([Ca2+]i) in vascular smooth muscle cells. Induction of c-fos is a critical early event in cell growth; expression of this gene is regulated by a number of signaling pathways including Ca2+. We wondered whether Ca2+ signaling plays a critical role in the induction of c-fos gene by α1-adrenergic receptors. Using stably transfected rat-1 fibroblasts, we confirmed that PE induced c-fos mRNA expression in a time- and dose-dependent manner, and also increased [Ca2+]i (measured with Fura-2 AM). These responses were blocked by the α1-adrenergic receptor antagonist doxazosin. Both intracellular Ca2+ chelation (using BAPTA/AM) and extracellular Ca2+ depletion (using EGTA) significantly inhibited PE-induced c-fosexpression by α1A and α1B receptors. Brief (1-min) stimulation of α1A and α1Breceptors with PE did not maximally induce c-fosexpression, suggesting that a sustained increase in [Ca2+]i due to Ca2+ influx is required. The calmodulin (CaM) antagonists, R24571, W7, and trifluoperazine, but not the CaM-dependent protein kinases inhibitor KN-62, significantly inhibited c-fos induction by α1A and α1B receptors. Neither inhibition of protein kinase C nor inhibition of adenylyl cyclase modified c-fos induction by PE. These results suggest that α1-adrenergic receptor-induced c-fosexpression in rat-1 cells is dependent on a Ca2+/CaM-associated pathway.
Footnotes
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Send reprint requests to: Brian B. Hoffman, M.D., Veterans Affairs Medical Center, Geriatrics Research, Education and Clinical Center 182B, 3801 Miranda Ave., Palo Alto, CA 94304. E-mail:bhoffman{at}leland.stanford.edu
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↵1 This study was supported in part by a grant (HL41315) from National Institutes of Health and the Research Service of the VA.
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↵2 Recipient, National Research Service Award (Institutional), and Fellowship for Careers in Clinical Pharmacology from the Pharmaceutical Research and Manufacturers of America (PhRMA) Foundation.
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↵3 Current address: University of Texas Health Science Center at San Antonio, Department of Pharmacology, San Antonio, TX 78284.
- Abbreviations:
- CaM
- calmodulin
- CaM kinase
- Ca2+/CaM-dependent kinases
- PKA
- protein kinase A
- PKC
- protein kinase C
- MAP kinase
- mitogen-activated protein kinase
- ERK
- extracellular stimulus response kinase
- CREB
- cAMP response element binding protein
- CRE
- cAMP response element
- SRE
- serum response element
- R24571
- calmidazolium chloride
- PMA
- phorbol 12-myristate 13-acetate
- HBSS
- Hanks’ balanced saline solution
- W7
- N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide
- DMEM
- Dulbecco’s modified Eagle’s medium
- BAPTA/AM
- 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester
- MBP
- myelin basic protein
- PKI
- protein kinase A inhibitory peptide
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- Received August 31, 1998.
- Accepted January 29, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
