X‐ROS Signaling in Cerebral Artery Smooth Muscle

Abstract

Mechanically induced generation of reactive oxygen species (ROS) by NADPH oxidase (NOX), referred to as X‐ROS, influences the contractility of cardiac and skeletal muscle, but the impact of this signaling pathway in smooth muscle has not been reported. Here we investigate the effect of X‐ROS on the excitability and contractility of cerebral artery smooth muscle cells (SMC). In these cells, spatially and temporally limited release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RyR) generate microdomains of high intracellular [Ca2+] known as Ca2+ sparks. Ca2+ sparks are functionally coupled to multiple large‐conductance Ca2+‐activated K+ (BK) channels and activate macroscopic Spontaneous Transient Outward Currents (STOCs) that cause membrane hyperpolarization and vasodilation. Several sites subject to redox modification are present on RyRs that influence activity. Therefore, we hypothesized that X‐ROS elevates Ca2+ spark and STOC activity, leading to membrane hyperpolarization and vasodilation. Experiments were carried out using cerebral pial arteries and freshly isolated cerebral artery SMC from C57/Bl6 mice. Ca2+ sparks were recorded from cerebral SMC loaded with the fast Ca2+ indicator fluo‐4AM using high‐speed, high‐resolution confocal microscopy. To determine if the production of O2− was required for generation of Ca2+ sparks, superoxide dismutase (SOD) was applied to the bath. Membrane permeable PEG‐SOD (100 U.ml−1, n = 5) nearly abolished Ca2+ spark activity. To further characterize this response, SMC were patch‐clamped using the amphotericin B perforated‐patch whole cell method to record STOCs. PEG‐SOD (100 U.ml−1, n = 6) attenuated STOC frequency, whereas non‐membrane permeable SOD (100 U.ml−1, n = 7) had no effect. The membrane permeable SOD mimetic TEMPO (1 mM, n = 5) also, attenuated STOC activity. Catalase (750 U.ml−1, n = 5) or membrane permeable PEG‐catalase (750 U.ml−1, n = 6) had no effect on STOC frequency, suggesting that H2O2 intermediates are not involved in this signaling pathway. Inhibition of NOX2 with apocynin (30 μM, n = 7) or gp91ds‐tat (1 μM, n = 6) greatly attenuated STOC frequency, whereas application of exogenous NADPH to increase NOX2 activity resulted in a concentration‐dependent increase in STOC frequency, with an EC50 of 61 nM (n = 3–8). These data indicate that intracellular generation of O2− by NOX2 is necessary for Ca2+ spark and STOC activity. Pressure myography experiments were performed to determine the functional significance of the X‐ROS pathway in SMC. Endothelium denuded posterior cerebral arteries were pressurized to 50 mmHg to generate spontaneous myogenic tone and then treated with the substrate for NOX, NADPH (10 μM). NADPH caused vasodilation that was reversed by the BK blocker paxilline (1 μM, n = 5), suggesting that NOX activity dilates cerebral arteries by activating BK channels. We conclude that generation of intracellular O2− by NOX2 stimulates Ca2+ spark and STOC activity, resulting in cerebral arteries dilation. These data highlight a novel ROS‐dependent signaling pathway that may critically influence cerebral blood flow regulation.Support or Funding InformationSupport: R01HL091905 (SE), AHA15POST2472002 (PWP).