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Negative feedback regulation of reactive oxygen species on AT1 receptor gene expression
G Nickenig1, K Strehlow, A T Bäumer
1Klinik III für Innere Medizin, Universität Köln, Joseph-Stelzmann Strasse 9, 50925 Köln, Germany. georg.nickenig@uni-koeln.de
Abstract:
Free radicals as well as the AT1 receptor are involved in the pathogenesis of cardiovascular disease. Both the intracellular mechanisms of AT1 receptor regulation and the effect of free radicals on AT1 receptor expression are currently unknown. This study investigates the role of free radicals in the modulation of AT1 receptor expression and in the angiotensin II-induced AT1 receptor regulation. AT1 receptor mRNA was assessed by Northern blotting and AT1 receptor density by radioligand binding assays, respectively, in vascular smooth muscle cells (VSMC). Free radical release was measured by confocal laser scanning microscopy. AT1 receptor mRNA transcription rate was determined by nuclear run-on assays and AT1 receptor mRNA half-life was measured under transcriptional blockade. Angiotensin II caused a time-dependent decrease of AT1 receptor mRNA expression in rat VSMC in culture (30+/-6% at 4 h with 100 nM angiotensin II). This was followed by a consistent decrease in AT1 receptor density. Angiotensin II caused release of reactive oxygen species in VSMC which was abolished by preincubation with 100 microM diphenylene iodonium (DPI). DPI inhibited partially the down-regulating effect of angiotensin II on the AT1 receptor. Incubation of VSMC with either hydrogen peroxide or xanthine/xanthine oxidase caused a dose-dependent decrease in AT1 receptor mRNA expression which was not mediated by a decreased rate of transcription but rather through destabilization of AT1 receptor mRNA. Experiments which included preincubation of VSMC with various intracellular inhibitors suggested that free radicals caused AT1 receptor downregulation through activation of p38-MAP kinase and intracellular release of calcium. However, angiotensin II-induced AT1 receptor expression was not inhibited by blockade of p38-MAP kinase activation or intracellular calcium release. Free radicals may at least in part mediate angiotensin II-induced AT1 receptor regulation through direct post-transcriptional effects on AT1 receptor mRNA expression which involves intracellular release of calcium and activation of p38-MAP kinase. These findings may help to clarify the intracellular mechanisms involved in AT1 receptor regulation and reveal a novel biological feature for reactive oxygen species.
Insights
Free radicals, or reactive oxygen species, reduce angiotensin II type 1 receptor (AT1 receptor) expression by destabilizing its mRNA. This occurs through intracellular calcium release and p38-MAP kinase activation, impacting cardiovascular disease mechanisms.
Area of Science:
- Cardiovascular Science
- Molecular Biology
- Cellular Signaling
Background:
- The angiotensin II type 1 receptor (AT1 receptor) is crucial in cardiovascular disease pathogenesis.
- Intracellular mechanisms regulating AT1 receptor and the impact of free radicals on its expression remain unclear.
Purpose of the Study:
- To investigate the role of free radicals in modulating AT1 receptor expression.
- To elucidate the mechanisms of angiotensin II-induced AT1 receptor regulation by free radicals.
Main Methods:
- Assessed AT1 receptor mRNA and density in vascular smooth muscle cells (VSMC) using Northern blotting and radioligand binding assays.
- Measured free radical release via confocal microscopy, and AT1 receptor mRNA transcription/half-life using nuclear run-on assays and transcriptional blockade.
- Utilized diphenylene iodonium (DPI), hydrogen peroxide, xanthine/xanthine oxidase, and intracellular inhibitors (p38-MAP kinase, calcium signaling).
Main Results:
- Angiotensin II decreased AT1 receptor mRNA and density in VSMC, associated with reactive oxygen species release.
- Free radicals (hydrogen peroxide, xanthine/xanthine oxidase) dose-dependently decreased AT1 receptor mRNA by destabilizing the mRNA, not affecting transcription rate.
- Free radical-induced downregulation involved p38-MAP kinase activation and intracellular calcium release, but these pathways were not essential for angiotensin II-induced AT1 receptor downregulation.
Conclusions:
- Free radicals contribute to angiotensin II-induced AT1 receptor regulation via post-transcriptional effects on mRNA stability.
- Intracellular calcium release and p38-MAP kinase activation are key mediators in free radical-induced AT1 receptor downregulation.
- Findings clarify AT1 receptor regulation mechanisms and highlight a novel role for reactive oxygen species in cardiovascular pathology.