Abstract 930: NADPH Oxidase 2 is Responsible for α1-Adrenergic Receptor-Dependent Reactive Oxygen Species Production in Adult Rat Ventricular Myocytes
Background: Five NAPDH oxidase (NOX) isoforms have been implicated in reactive oxygen species (ROS) generation in a cell type- and agonist-specific manner. We previously showed that α1-adrenoreceptor (α1-AR) stimulation in cultured adult rat ventricular myocytes (ARVM) leads to NADPH-dependent ROS-production, suggesting that a NOX may be responsible. The goal of this project was to determine whether NOX mediates α1-AR-stimulated ROS production in ARVM, and if so, which isoform(s) is responsible.
Methods and Results: ROS production, assessed by dichlorofluoresceine (DCF) fluorescence, was increased by 51±7 % in ARVM subjected to α1-AR stimulation (norepinephrine 1μM + propranolol 2 μM; 30 min; p=0.002, n=3/group). Apocynin (Apo, 50 μM), a broad-spectrum NOX inhibitor, prevented α1-AR-mediated ROS generation (+19±8% vs Apo-treated/α1-AR-unstimulated cells, n=4, NS), suggesting a role for NOX. Expression of NOX1, 2 and 4, as well as regulatory proteins p22phox, p47phox, Rac1 and Rac2, but not NOX3, was detected by RT-PCR in ARVM. Neither the inhibitory Nox4-tat peptide (+56±7% vs NOX4-tat-treated/α1-AR-unstimulated cells) nor adenovirus-mediated overexpression of NOX4 dominant-negative protein (+84±17% vs NOX4DN-infected/α1-AR-unstimulated cells) reduced α1-AR-induced DCF fluorescence increase (n=4/ group, both p<0.01 vs α1-AR-unstimulated cells). In contrast, adenovirus-mediated overexpression of dominant negative p47phox, p67phox and Rac1 proteins prevented α1-AR-mediated ROS production, suggesting that the responsible NOX requires each of these proteins. NOX2-tat inhibitory peptide (+18.4±7.3% vs NOX2-tat-treated/α1-AR-unstimulated cells, n=6, NS), but not NOX1-tat inhibitory peptide (+51±8% vs NOX1-tat-treated/α1-AR-unstimulated cells, n=3, p<0.01), prevented α1-AR-mediated ROS production.
Conclusion: The NOX2 complex, including the catalytic subunit gp91phox, and the regulatory proteins p47phox, p67phox and Rac1, is directly responsible for α1-adrenergic-receptor induced ROS production in adult rat ventricular myocytes.