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Function and Regulation of Pannexin 1 Channels in the Vascular Endothelium

Lohman, Alexander
Format
Thesis/Dissertation; Online
Author
Lohman, Alexander
Advisor
Isakson, Brant
Abstract
The nucleotide adenosine 5’-triphosphate (ATP) has classically been considered the cell’s primary energy currency; however, a novel role for ATP as an extracellular autocrine/paracrine signaling molecule has evolved over the past century. Purinergic signaling is now known to regulate a plethora of physiological and pathophysiological processes in almost every organ system. In the vasculature, ATP and its metabolites elicit dual control over blood vessel tone and tissue perfusion, and purinergic signaling events have been implicated in vascular pathologies including atherosclerosis and inflammation. While the importance of extracellular ATP in the vascular system is well recognized, the mechanism(s) mediating the regulated release of the purine from vascular cells are less well understood and are a key target of current investigation. One such ATP-liberation mechanism has been ascribed to the recently identified pannexin (Panx) channels, namely Panx1. Initially, we characterized the expression and localization profiles of Panx isoforms across the systemic vasculature, identifying predominant representation by the Panx1 isoform in both smooth muscle (SMC) and endothelial cells (EC) comprising the blood vessel wall, with more heterogeneous expression profiles observed in specialized vascular systems including the heart, lung and kidney. In particular, the Panx1 isoform is highly expressed in ECs regardless of blood vessel type or size, while Panx1 expression is limited to SMCs of small arteries and arterioles. Panx1 forms hexameric channels in the plasma membrane of ECs, functioning to release ATP in response to a number of stimuli. However, prolonged Panx1 channel activity is extremely detrimental to cell viability. Here we report a novel negative regulatory mechanism that may govern the activity of Panx1 channels in ECs, by which the bioactive gas nitric oxide (NO) covalently modifies two cysteine (Cys) residues in the channel by a process termed S-nitrosylation. Targeted S-nitrosylation of both Cys40 in the channel’s predicted pore lining region and Cys346 in the C-terminal tail are required for Panx1 channel inhibition by NO. Finally, we present new data placing EC Panx1 channels at the center of the acute vascular inflammatory response. Extracellular ATP is a potent pro-inflammatory mediator where it regulates interactions between circulating inflammatory cells and the vascular endothelium, primarily in the venous circulation. Venous EC activation by the pro-inflammatory cytokine Tumor Necrosis Factor alpha (TNFα) promotes the recruitment and infiltration of circulating leukocytes to local sites of tissue injury or infection. We report that activation of venous EC TNFα Receptor Type 1 promotes Panx1 channel activation and ATP release into the vessel lumen through a signaling cascade dependent on Src Family Kinases. Pharmacological inhibition and targeted genetic deletion of Panx1 in the venous endothelium essentially blocked leukocyte-EC interactions in a model of acute vascular inflammation. Taken together, we identify EC Panx1 channels as novel regulators of vascular inflammation, functionally integrating inflammatory and purinergic signaling to promote proper homing of inflammatory cells throughout the body.
Language
English
Published
University of Virginia, Department of Molecular Physiology and Biological Physics, PHD, 2014
Published Date
2014-07-25
Degree
PHD
Collection
Libra ETD Repository
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