Arteriolar vascular smooth muscle cells (VSMCs) are mechanosensitive, constricting to elevations in transmural pressure (P TM ). The goal of the present study was to determine using mouse isolated tail arterioles and arteries whether oxidant signaling regulates this myogenic response. In response to P TM elevation, VSMCs of arterioles but not arteries generated constriction and increased reactive oxygen species (ROS) activity (using the H 2 O 2 -sensitive probe dichlorodihydrofluorescein). Arterioles had increased expression of NADPH oxidase components compared with arteries. Inhibition of NADPH oxidase, using mice with targeted impairment of enzyme components (p47 phox or rac1) or diphenyleneiodonium, prevented the pressure-induced generation of ROS. When ROS activity was inhibited, either by inhibiting NADPH oxidase or with N-acetylcysteine, the myogenic constriction was abolished. The myogenic constriction was also inhibited by catalase, which inactivates H 2 O 2 , but was unaffected by a cell-permeant mimic of superoxide dismutase (MnTMPyP). ␣ 1 -Adrenergic constriction was not associated with altered ROS activity and was not affected by inhibition of NADPH oxidase or ROS. Exogenous H 2 O 2 constricted VSMCs of arterioles but not arteries. Thus, NADPH oxidase and ROS, in particular H 2 O 2 , contribute to the myogenic response of arteriolar VSMCs. V ascular smooth muscle cells (VSMCs) of arterioles, but not arteries, are mechanosensitive, constricting to elevations in transmural pressure (P TM ). This myogenic response contributes to blood flow autoregulation and the establishment of basal vascular tone. 1 The response is an inherent property of arteriolar VSMCs involving calcium-dependent actin/myosin interaction, but the more proximal signaling components have not been clearly defined. 1 In cultured cells, mechanical stress initiates integrin-dependent activation of rho GTPases (rho, rac1, and CDC42), leading to reorganization of the cytoskeleton. 2,3 Cytoskeleton reorganization by rac1 is mediated by NADPH oxidase and generation of reactive oxygen species (ROS). 4 This enzyme complex, comprising Nox1, p47 phox , p67 phox , p22 phox , and rac1, is a key signaling system in cultured, noncontractile VSMCs. 5,6 The aim of the present study was to determine whether the rac1/NADPH oxidase/ROS signaling pathway regulates the myogenic response of arteriolar VSMCs.
Materials and Methods
Vasomotor ResponsesMouse-tail arterioles and arteries were cannulated in a microperfusion chamber (Living Systems) and studied in the absence of flow as described. 7 Unless stated otherwise, arterioles with intact endothelium were analyzed. Involvement of animals in the study was approved by the Ohio State University Animal Care and Use Committee.
ROS DeterminationEndothelium-denuded vessels 7 were incubated with the H 2 O 2 -sensitive probe 5-(and 6)-chloromethyl-2Ј,7Ј-dichlorodihydro-fluorescein diacetate (DCF), 5 g/mL, for 30 minutes (37°C, P TM of 10 mm Hg). Because activation of DCF fluorescence is irreversible, fluorescent im...
