Mechanisms of Intracellular NAMPT-regulated GSNOR in Vessel Wall

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Abstract The regulation of resistance arterial tone involves communication between vascular smooth muscle and endothelium, which is tightly controlled by an intricate, but yet to be fully defined, cell signaling processes. Recently, we made the discovery that S-nitrosylation/denitrosylation, the addition or removal of a nitric oxide group from a cysteine-thiol side chain, serves as an important post-translational modification on connexin 43 gap junction (GJ) proteins, and that this modification is associated with control of resistance arterial tone. Regulation of connexin 43 nitrosylation appeared to be predominant at the myoendothelial junction (MEJ), the point where endothelial cells and smooth muscle cells make contact in resistance arteries. At the MEJ, endothelial nitric oxide synthase (eNOS), and the denitrosylase S-nitrosoglutathione reductase (GSNOR), work in concert to modulate the permeability of GJs. The mechanisms regulating eNOS activity have been well characterized, however the molecular mechanisms regulating GSNOR activity remain poorly understood. To identify enriched proteins at the MEJ capable of regulating GSNOR activity, we recently performed an in vitro MEJ proteomic screen. From this analysis, we found enriched expression of nicotinamide phoshoribosyltransferase (NAMPT), a rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD) biosynthesis pathway. The localized protein expression of intracellular NAMPT at the MEJ suggested to us that it is critical for the regulation of NAD levels, which are known to modulate GSNOR activity and thus might control heterocellular communication in the vessel wall. In our pilot studies, we explored key elements of this concept by showing that intracellular NAMPT can regulate GSNOR activity and resistance arterial tone. Based on these observations we formulated the central hypothesis that vascular resistance and thus, systemic blood pressure control is mediated through a localized NAMPT-regulated GSNOR mechanism. We will test this hypothesis using three specific aims: AIM 1 will test whether NAMPT regulates GSNOR activity and heterocellular communication in vitro, AIM 2 will determine if NAMPT is critical in the regulation of resistance arterial tone, AIM 3 will elucidate how cell-type specific modulation of NAMPT expression in endothelium or smooth muscle modifies the responses to vasoconstrictors or vasodilators in resistance arteries. Our results will impact our understanding of these enzymes in blood pressure control and provide a framework to determine whether dysfunctions in the expression and/or activity of NAMPT and GSNOR contribute to cardiovascular diseases including hypertension.