Increased resistance to sinusoidal blood flow is an important component of early as well as advanced portal hypertension. This results from an imbalance in intrahepatic vasoconstrictor and vasodilator molecules, the latter including nitric oxide (NO). Our laboratory has been on the cutting edge of advances in understanding the molecular basis for the imbalance in NO that occurs after liver injury. Seminal among our discoveries is that there is a (dramatic and remarkable) reduction in endothelial NO synthase (eNOS) dependent NO release by sinusoidal endothelial cells (SECs) after liver injury. Further, this “endothelialopathy” is a critical feature of portal hypertension, especially early in disease. Our focus has been on understanding the mechanism underlying this defect. Preliminary data presented in the current application has identified highly novel post-translational defects in eNOS in SECs after liver injury, including reduced phosphorylation of eNOS caused by abnormalities in molecules that form a macromolecular regulatory complex that controls eNOS function (including the following G protein coupled receptor (GPCR) regulatory proteins: β-arrestins, GIT1 and now β-PIX). Additionally, many of these partners are regulated during liver injury. In the current project, we will test the highly focused and innovative central hypothesis that the GIT1/β-PIX complex and its partners play a fundamental role in regulating eNOS function. Therefore, overarching goals of this new project are twofold. First, we wish to uncover fundamental basic mechanisms that regulate eNOS function. Secondly, with a future objective being to translate our work to humans with liver disease, we wish to validate the importance of the proposed signaling pathways in vivo. Therefore, our specific aims are as follows: We will (1) determine how β-PIX, as a GIT1 binding partner, exerts a dual role in post-translational regulation of eNOS activity in normal versus injured SECs, and (2) we will characterize the function of the GIT1/β-PIX complex partner, paxillin, in regulating eNOS function through interaction with β-PIX and GIT1 after liver injury. The proposed experiments will uncover novel mechanistic aspects of eNOS function and as such have fundamental therapeutic implications not only for patients with liver disease and portal hypertension, but also for those with other vascular disorders.