Negative feedback regulation of reactive oxygen species on AT1 receptor gene expression

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.

Figure 1

Quantification of Northern hybridization signal intensity. Time course of the AT1 receptor as well as GAPDH mRNA in the presence of either vehicle (A) or 100 nM Angiotensin II (B). Northern hybridizations were performed as described in Experimental Procedures. Each point represents the relative hybridization signal normalized to the 0 h treatment with vehicle (100%) from five separate experiments±s.e. ^*P<0.05.

Figure 2

Effect of angiotensin II on free radical release in VSMC. (A) Representative microscopic laserscan of VSMC incubated for 3 h with vehicle (A), 100 μM DPI (B), 100 nM angiotensin II, or DPI and angiotensin II (D). Free radical release is visualized through DCF fluorescence. (B) Quantification of angiotensin II-induced release of free radicals. VSMC were incubated for 3 h with vehicle (control), 100 μM DPI, 100 nM angiotensin II (AngII), or DPI and angiotensin II (D). Free radical release is visualized through DCF fluorescence. means±s.e. ^*P<0.05.

Figure 3

Effect of DPI on angiotensin II-induced AT1 receptor down-regulation. VSMC were incubated for 4 h with vehicle (control), 100 nM angiotensin II (AngII), 100 μM DPI, or angiotensin II and DPI. AT1 receptor and GAPDH mRNA were quantified by Northern Blots. Densitometric analysis of three separate experiments±s.e. ^*P<0.05 vehicle versus angiotensin II, ^**P<0.05 angiotensin II versus DPI+angiotensin II.

Figure 4

Effect of xanthine oxidase and hydrogen peroxide on AT1 Receptor mRNA. VSMC were incubated for 24 h with vehicle, 0.1–100 μM hydrogen peroxide (A) or 0–1.6 μU ml⁻¹ xanthine oxidase (B). AT1 receptor and GAPDH mRNA were quantified by Northern Blots. Densitometric analysis of three separate experiments. mean±s.e. ^*P<0.05 VSMC were incubated for 0–24 h with 0.1–100 μM hydrogen peroxide (C). Representative Northern Blot showing AT1 receptor and 18S and 28S mRNA expression.

Figure 5

Effect of hydrogen peroxide on AT1 receptor protein expression. VSMC were incubated for 24 h with 100 μM hydrogen peroxide. Proteins were isolated and Western blots were performed to quantify AT1 receptor protein. Representative Western blot and densitometric analysis. mean±s.e. ^*P<0.05.

Figure 6

Effect of hydrogen peroxide on de novo mRNA synthesis in VSMC. VSMC were incubated with vehicle, 100 μM hydrogen peroxide, or 20 ng ml⁻¹ EGF. Nuclei were isolated and nuclear run-on assays were performed. Autoradiograms were quantified by laser densitometry. Each point represents three separate experiments±s.e. ^*P<0.05.

Figure 7

Effect of hydrogen peroxide on AT1 receptor mRNA stability. VSMC were incubated for 12 h with either 100 μM hydrogen peroxide or vehicle before 50 μg ml⁻¹ DRB was added. Total RNA was isolated at the indicated time points and AT1 receptor mRNA was quantified by Northern analysis. Each point represents data from five separate experiments±s.e. ^*P<0.05.

Figure 8

Second messenger involved in hydrogen peroxide-induced AT1 receptor mRNA down-regulation. Cells were incubated for 24 h with 100 μM hydrogen peroxide in the presence of either 1 μM PD98059 (PD), 1 μM SB203580 (SB), 1 μM herbimycin (Herbi) or 20 μM Bis-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (Maptam). Inhibitors were added 30 min prior to hydrogen peroxide. AT1 receptor and GAPDH mRNA was assessed by Northern analysis. Each point represents data from five separate experiments±s.e. ^*P<0.05 control versus H₂O₂. ^**P<0.05 H₂O₂ versus H₂O₂+MAPTAM or SB.

Figure 9

Intracellular calcium release in vascular smooth muscle cells. Confluent cells were stimulated with 1–100 μM hydrogen peroxide before the intracellular calcium concentration was monitored with confocal microscopy. Representative graph generated from cells stimulated with 100 μM hydrogen peroxide (A) and summarized data (B) of stimulation with increasing concentrations of hydrogen peroxide.