β-Adrenergic Stimulation of Rat Cardiac Fibroblasts Enhances Induction of Nitric-Oxide Synthase by Interleukin-1β via Message Stabilization
- 1Biomedical Sciences Graduate Program (Å.B.G.), Departments of Pharmacology and Medicine (L.L.B.), University of California at San Diego, La Jolla, California
Abstract
We have investigated factors modulating expression of inducible NO synthase (iNOS) in isolated adult rat cardiac fibroblasts. Treatment of cardiac fibroblasts with interleukin-1β (IL-1β) promotes induction of iNOS mRNA and protein and production of NO. Simultaneous incubation of cells with isoproterenol enhances the response to IL-1β, even though isoproterenol alone is without effect.N G-nitro-l-arginine methyl ester inhibits the effect of isoproterenol + IL-1β on NO production. β2-Adrenergic receptors appear to mediate this effect of isoproterenol. Reverse transcriptase-polymerase chain reaction analyses show that β2-receptor mRNA is the predominant β-receptor message; in pharmacologic studies, ICI-118,551 significantly antagonizes isoproterenol-stimulated cyclic AMP production whereas CGP20712A does not. Dibutyryl-cyclic AMP and forskolin mimic the synergistic effect of isoproterenol on IL-1β-induced NO production; H-89, a cyclic AMP-dependent protein kinase (PKA) inhibitor, antagonizes the enhancing effect of isoproterenol. Nuclear run-off experiments indicate that enhancement of iNOS by isoproterenol does not occur at the level of transcription. Message stability studies demonstrate that isoproterenol increases the half-life of iNOS mRNA from 1.0 to 1.9 h; this change is sufficient to account for the observed augmentation of iNOS mRNA and protein. Thus, cardiac fibroblasts produce significant amounts of NO in response to IL-1β via induction of iNOS; β-adrenergic stimulation enhances the IL-1β effect by stabilizing the iNOS message. These data suggest that cardiac fibroblasts could participate in a paracrine mechanism whereby the direct positive inotropic effect of β1-adrenergic stimulation of myocytes is opposed by β2-adrenergic enhancement of NO production, a negative inotropic event, in neighboring fibroblasts.
Footnotes
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Send reprint requests to: Åsa B. Gustafsson, Department of Pharmacology 0636, UCSD School of Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0636. E-mail: agustafsson{at}ucsd.edu.
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This work was supported by National Institutes of Health Grants HL41307 and GM07752 and a predoctoral fellowship from the American Heart Association, Western States Affiliates (to Å.B.G).
- Abbreviations:
- NO
- nitric oxide
- iNOS
- inducible nitric-oxide synthase
- IL
- interleukin
- TNF
- tumor necrosis factor
- IFN
- interferon
- l-NAME
- NG-nitro-l-arginine methyl ester
- PKA
- cyclic AMP-dependent protein kinase
- DMEM
- Dulbecco's modified Eagle's medium
- BSA
- bovine serum albumin
- Iso
- isoproterenol
- DRB
- 5,6-dichloro-1-β-ribofuranosyl benzimidazole
- PCR
- polymerase chain reaction
- RT-PCR
- reverse transcriptase-PCR
- IBMX
- isobutylmethylxanthine
- db-cAMP
- dibutyryl-cAMP
- EPI
- epinephrine
- NE
- norepinephrine
- bp
- base pair
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- Received January 31, 2000.
- Accepted September 19, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
