Abstract 152: Urotensin-II Activates Rac-1 And ROS Production In Pulmonary Artery Smooth Muscle Cells Involving GPR14 And GαI3 Proteins
Human urotensin II (hU-II) has recently been described as a potent vasoactive peptide associated with remodelling processes within the vasculature, thus contributing to various cardiovascular diseases including pulmonary hypertension. hU-II acts via the G-protein coupled receptor GPR14. However, the mechanisms mediating GPR14 signalling remain unclear. ROS have been shown to act as signalling molecules in vascular cells, and NADPH oxidases which are activated by Rac-1, are important sources of ROS generation. Therefore, we investigated whether hU-II and GPR14 can activate Rac-1 and NADPH oxidase-mediated ROS production in pulmonary artery smooth muscle cells (PASMC). Urotensin-II activated Rac-1 within 15 seconds and this response was abolished by treatment of PASMC with the GPR14 antagonist urantide. In line, urotensin-II induced a rapid increase in ROS levels within 5 minutes of application, which was prevented by urantide or transfection of a dominant-negative mutant of Rac-1 (RacT17N). Furthermore, in cells overexpressing GPR14 or constitutively active Rac-1 (RacG12V), hU-II-stimulated ROS generation was further enhanced. Interestingly, in the presence of Pertussis toxin, hU-II-induced Rac-1 activation and ROS generation were abolished, indicating coupling of GPR14 to Gαi proteins. Indeed, transfection of dominant-negative G αi3 completely diminished hU-II-stimulated ROS generation in PASMC. Similar effects were observed upon depletion of the NADPH oxidase subunit NOX4. Furthermore, hU-II, RacG12V and NOX4 stimulated proliferation of PASMC, whereas antioxidants, RacT17N or NOX4 depletion had opposite effects.These results show that hU-II is a potent activator of Rac-1 leading to rapid activation of NADPH oxidase and ROS generation involving GPR14 and Gαi3 proteins, and subsequent stimulation of PASMC proliferation. Thus, activation of NADPH oxidase by GPR14 and Rac-1 provides a novel mechanism how hU-II can promote vascular remodelling processes and may provide a novel therapeutic target for pulmonary vasculopathies associated with pulmonary hypertension.