Abstract
Activation of the endogenous α1-adrenergic receptor (AR) associated with human aortic smooth muscle cells resulted in a dose- and time-dependent increase in the levels of mitochondrial reactive oxygen species (ROS). ROS increases were apparent within 10 min and maximal after 45 min. Prolonged activation (>4h) of the α1-AR resulted in smooth muscle cell apoptosis. Both the increase in ROS and apoptotic cell death were blocked by the nonselective α1-AR antagonist prazosin as well as the selective α1D-AR antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7, 9-dione (BMY 7378). Increases in ROS and apoptosis produced by α1-AR activation were also blocked by the p38 mitogen-activated protein kinase inhibitor 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB 202190) and the NAPDH oxidase inhibitor apocynin. The extracellular signal-regulated kinase 1/2 inhibitor 2′-amino-3′-methoxyflavone (PD 98059) or the c-Jun NH2-terminal kinase inhibitor 1, 9-pyrazoloanthrone anthra(1, 9-cd)pyrazol-6(2H)-one (SP 600125) was without effect on increases in ROS levels or apoptosis. Pifithrin-α, an inhibitor of the tumor suppressor protein p53, had no effect on ROS generation but did block α1D-AR-induced apoptosis. Activation of the α1D-AR resulted in translocation of p53 to the mitochondria. The mitochondrial translocation of p53 was blocked by prazosin, BMY 7378, apocynin, SB 202190, and pifithrin-α. Apoptosis was also blocked by small interfering RNA directed against p53. These data show that the α1D-AR is coupled to the generation of mitochondrial ROS by a pathway involving p38 and NADPH oxidase. Sustained activation of the α1D-AR results in smooth muscle cell apoptosis in a pathway that involves the tumor suppressor protein p53 and the mitochondrial translocation of p53. The data also provide evidence of a linkage between the α1D-AR and p53.
Footnotes
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This study was supported by National Institutes of Health grants HL38120-14 (to M.T.P.) and CA73599 and CA94853 (to D.R.St.C.).
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M.L.G.-C. and J.L.S. contributed equally to this work.
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ABBREVIATIONS: AR, adrenergic receptor; ROS, reactive oxygen species; siRNA, small interfering RNA; BMY 7378, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7, 9-dione; WB 4101, 2-(2, 6-dimethoxyphenoxyethyl)aminomethyl-1, 4-benzodioxane hydrochloride; MAP, mitogen-activated protein; SB 202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole; ERK, extracellular signal-regulated kinase; PD 98059, 2′-amino-3′-methoxyflavone; JNK, c-Jun NH2-terminal kinase; SP 600125, 9-pyrazoloanthrone anthra(1, 9-cd)-pyrazol-6(2H)-one; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; ANOVA, analysis of variance.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received April 23, 2008.
- Accepted July 14, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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