(Verbatim from the application): In cultured cells, mechanical strain induces a change in the actin cytoskeleton, mediated in part by the small Gprotein, rac. Rac stimulates actin polymerization, via NADPH oxidase-dependent increase in reactive oxygen species (ROS), which stimulate uncapping of actin filaments. The role of rac in contractile smooth muscle cells (SM) is unknown. We demonstrate that a signaling pathway(s), involving rac, NADPH oxidase, ROS and actin polymerization, plays a key role in the myogenic response, the arteriolar SM constriction caused by elevation in transmural pressure. In mouse arterioles, the myogenic response was: i) selectively abolished by antioxidants or inhibition of NADPH oxidase, ii) depressed in transgenic mice expressing a dominant-negative mutant of rac (RAC-DN) in SM, iii) increased in transgenic mice expressing a constitutively-active rac mutant (RAC-CA) in SM, and iv) associated with increased production of ROS that was absent in RAC-DN arterioles. Inhibition of actin polymerization by cytochalasin D selectively inhibited the myogenic response. Based on these experiments, and on current models of smooth muscle contraction, we propose that the myogenic response involves two distinct signaling pathways. One involves the conventional pathway, namely calcium influx, calcium-calmodulin dependent activation of myosin light chain kinase and phosphorylation of myosin light chain. The other pathway involves activation of raci, NADPH oxidase, elevation of ROS production and stimulation of actin polymerization. This latter pathway enables the VSM to withstand elevated pressure and allows the myosin-based system to constrict the artery. Furthermore, since amplification of this response in the RAC-CA mouse was associated with hypertension, we propose that dysregulation of this pathway may contribute to altered vascular function in this disease process.
Three specific aims are proposed to analyze the physiological and pathophysiological regulation and role of ROS in arterioles. Experiments will assess the stimuli responsible for ROS production, the mechanisms underlying ROS-induced changes in vasoconstriction and the regulation of this signaling pathway in hypertension.
