Mechanisms of NAMPT-stimulated nitric oxide release at the myoendothelial junctio

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DESCRIPTION (provided by applicant): Mechanisms of NAMPT-stimulated nitric oxide release at the myoendothelial junction Communication between endothelial cells (ECs) and smooth muscle cells (SMCs) is imperative for blood pressure regulation and vasomotor responses in the resistance vasculature. The myoendothelial junction (MEJ) is unique to the resistance vasculature and is hypothesized to be a highly organized cell-signaling microdomain that can regulate crosstalk between ECs and SMCs. Recently, caveolae were identified at the MEJ, providing circumstantial evidence that unique signaling events at this juncture. Additionally, numerous reports have demonstrated that caveolae regulate endothelial nitric oxide synthase (eNOS) transport and activity. These discoveries led us to hypothesize that eNOS synthase may polarize to the MEJ and is compartmentalized to promote highly localized cell signaling. Our preliminary results confirm that eNOS localizes to the MEJ and is selectively phosphorylated at the MEJ in vitro. To identify potential activators of eNOS, we performed a proteomic analysis of proteins that were enriched at the MEJ and found nicotinamide phoshoribosyltransferase (NAMPT). Importantly, NAMPT is a recently characterized adipokine that can stimulate eNOS phosphorylation, nitric oxide release, and endothelium dependent vasodialation. The mechanisms by which NAMPT can activate eNOS remain unclear, however it has been suggested that the insulin receptor- 2, which also polarizes to the MEJ, plays an intermediary role. Therefore, the aim of this proposal is to identify mechanisms of NAMPT regulated nitric oxide release at the MEJ, with a particular focus on understanding localized nitric oxide signaling at the MEJ Specifically, the goals of this proposal are to; 1.) define localized expression of eNOS, IR-2, and NAMPT at the MEJ and post-translational modifications, 2.) understand the mechanism of NAMPT stimulated eNOS signaling at the MEJ, 3) generate a NAMPT/EC specific IR-2 knockout animal to determine in vivo mechanisms of blood pressure and vasomotion regulation. This research plan has the additional benefit of providing an investigational framework upon which pharmacological studies of compartmentalized NAMPT/eNOS signaling at the MEJ may be performed, with the long term aim of preventing and or reversing cardiovascular related diseases. PUBLIC HEALTH RELEVANCE: Within the resistance vasculature endothelial cells and vascular smooth muscle cells are coupled in part by myoendothelial junctions (MEJ). The focus of this proposal entails the compartmentalization and function of endothelial nitric oxide synthase (eNOS) and nicotinamide phoshoribosyltransferase (NAMPT) at the MEJ. We will attempt to elucidate the interaction between NAMPT and eNOS as well as the potential implications this may have on the regulation of blood pressure and vasomotion.