Abstract

Abstract: DESCRIPTION (provided by applicant): Inflammatory mediators induce gaps between endothelial cells that are sufficient to increase tissue edema and facilitate transmigration of circulating blood elements to sites of injury. A rise in cytosolic Ca²⁺ ([Ca²⁺]i) triggers the cytoskeletal reorganization, decrease in cell adhesion, and increase in tension that causes gap formation. This [Ca²⁺]i response to inflammatory mediators is broadly characterized by two phases, including Ca²⁺ release from intracellular stores and Ca²⁺ entry across the cell membrane. These phases are inter-related since Ca²⁺ release stimulates Ca²⁺ entry in a process generally referred to as store operated Ca2+ entry. It is Ca²⁺ entry and not Ca²⁺ release that promotes endothelial cell gap formation, although he ion channels that mediate this rise in [Ca²⁺]i are unknown. Emerging evidence indicates that while inflammatory agonists activate both Ca²⁺ selective and non-selective cation channels, it is a Ca²⁺ selective channel that is uniquely coupled to loss of endothelial cell barrier integrity. Moreover, our preliminary data suggest two subunits of the transient receptor potential gene family, so called Trp1 and Trp4, combine to form the molecular basis of this Ca²⁺ selective channel Trp1 and Trp4 appear to interact directly with the spectrin membrane skeleton and consequently activation of a Trp1 and Trp4 channel provides a Ca²⁺ source immediately adjacent to cytoskeletal structures that regulate endothelial cell shape. This proposal therefore tests the OVERALL HYPOTHESIS: that activation of a Trp1/r-dependent store operated Ca²⁺ entry pathway promotes reorganization of the membrane skeleton necessary to increase endothelial permeability. Specific Aim test the related hypotheses that: 1) Trp1 and Trp4 combine to form a Ca²⁺-selective store operated channel in endothelial cells: 2) Activation of a Trp1/4 channel requires its direct interaction with the spectrin membrane skeleton 3) Ca²⁺ entry through a Trp1/4 channel disrupts the spectrin-actin interaction and reorganizes f-actin important for increased permeability. Proposed studies are significant because they may reveal a/the ion channel that specifically regulated endothelial cell barrier function. If true, then pharmacological inhibitors of this channel could be developed as a novel anti-inflammatory therapy.

Thesaurus Terms:
calcium channel, calcium flux, cell morphology, protein protein interaction, vascular endothelium permeability, actin, cell adhesion, cytoskeleton, gap junction, respiratory epithelium, spectrin, vascular endothelium, vasoconstriction, electrophysiology, laboratory rat, tissue /cell culture