Non-cardiogenic pulmonary edema is a significant clinical problem for which there is no effective pharmacological therapy. Disruption of the pulmonary endothelial cell barrier is an initiating event that increases permeability and reduces blood oxygenation in this disease. Experimental models suggest neuro-humoral inflammatory mediators increase permeability at least partly by elevating cytosolic Ca2+ ([Ca2+]i) in endothelial cells, and that Ca2+ entry across the cell membrane is a required component of the permeability response. Despite this significance, mechanisms governing Ca2+ entry are poorly understood and to date there are no Ca2+ channels known to mediate Ca2+ entry. Emerging data support the idea that endothelial cell Ca2+ entry occurs following generation of inositol 1,4,5-trisphosphate which depletes intracellular Ca2+ stores. In this manner intracellular Ca2+ release induces Ca2+ entry through a process termed store operated Ca2+ entry. Recently, a store operated Ca2+ entry channel in the Drosophila melanogaster retina was discovered and called transient receptor potential or trp. Human homologs of this channel have been identified, including Htrp-1. Our preliminary data suggest Htrp-1 is present in pulmonary endothelial cells and may contribute to store operated Ca2+ entry. Moreover, activation of the putative Htrp-1 channel appears sufficient to change endothelial cell shape and promote permeability. This proposal therefore tests the overall hypothesis that agonist-evoked activation of an Htrp-1 Ca2+ entry channel is an important component of increased permeability.
Specific Aims test the related hypotheses that: (i) pulmonary endothelial cells express and Htrp-1 channel that regulates store operated Ca2+ entry, and (ii) activation of an Htrp-1 mediated Ca2+ current contributes to pulmonary endothelial cell contraction, reorganization of the F-actin cytoskeleton, generation of paracellular gaps and increased permeability. The proposed studies are significant since they test the role of a specific Ca2+ channel, Htrp-1, on control of endothelial [Ca2+]i and permeability. Completion of these studies may provide evidence of a Ca2+ channel that mediates Ca2+ entry in endothelial cells and is linked to control of cell shape and permeability.
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