NADPH Oxidase 1 Mediates Acute Blood Pressure Response to Angiotensin II by Contributing to Calcium Influx in Vascular Smooth Muscle Cells

Abstract

Background: Reactive oxygen species (ROS) and calcium ions (Ca2+) are among the major effectors of Ang II (angiotensin II) in vascular smooth muscle cells. ROS are related to Ca2+ signaling or contraction induced by Ang II, but little is known about their detailed functions. Here, NOX (NADPH oxidase), a major ROS source responsive to Ang II, was investigated regarding its contribution to Ca2+ signaling. Methods: Vascular smooth muscle cells were primary cultured from rat aorta. Ca2+ and ROS were monitored mainly using fura-2 and HyPer family probes, respectively. Signals activating NOX were examined with relevant pharmacological inhibitors and genetic manipulation techniques. Results: Ang II-induced ROS generation was found to be biphasic: the first phase of ROS production, which was mainly mediated by NOX1, was small and transient, preceding a rise in Ca2+, and the second phase of ROS generation, mediated by NOX1 and NOX4, was slow but sizeable, continuing over tens of minutes. NOX1-derived superoxide in the first phase is required for Ca2+ influx through nonselective cation channels. AT1R (Ang II type 1 receptor)-G(beta gamma)-PI3K(gamma) (phosphoinositide 3-kinase gamma) signaling pathway was responsible for the rapid activation of NOX1 in the first phase, while in the second phase, NOX1 was further activated by a separate AT1R-G alpha(q/11)-PLC (phospholipase C)-PKC beta (protein kinase C beta) signaling axis. Consistent with these observations, aortas from NOX1-knockout mice exhibited reduced contractility in response to Ang II, and thus the acute pressor response to Ang II was also attenuated in NOX1-knockout mice. Conclusions: NOX1 mediates Ca2+ signal generation and thereby contributes to vascular contraction and blood pressure elevation by Ang II.