The overall objective of this renewal application is to test the hypothesis that the activation of Trp channel-mediated Ca2+ entry in endothelial cells increases lung microvascular permeability and promotes lung edema. We have shown that thrombin activation of Proteinase-Activated Receptor-1 (PAR-1) expressed on endothelial cell surface induces an increase in intracellular Ca2+, which is critical in the mechanism of increased endothelial permeability. The increase in intracellular Ca2+ concentration ([Ca2+]i) is dependent on both Ca2+ store depletion and the Ca2+ store depletion-mediated Ca2+ influx. We showed that the prevention of Ca2+ influx drastically reduced the thrombin-induced increase in endothelial permeability. We also show that the Ca2+ influx due to store depletion occurs through activation of the transient receptor potential (Trp) gene family of channels expressed in endothelial cells. Our supporting data show that Trp1 (Trp gene homologue) is predominantly expressed in human endothelial cells and increased expression of Trp1 augments the Ca2+ influx in endothelial cells. Further, we show that Src kinase activation plays an important role in the regulation of Ca2+ influx via Trp channels. Deletion of Trp4 gene (predominant Trp isoform expressed in mouse endothelial cells) in mouse impairs thrombin-induced increase in lung microvascular permeability. The association of Trp1 with caveolin-1 (the principal protein of caveolae) in response to thrombin is required for the Ca2+ influx in endothelial cells. We also show that the inflammatory cytokine, tumor necrosis factor-alpha (TNFa) increases Trp1 expression in endothelial cells. Based on these supporting data, we propose to (i) determine the role of Src kinase signaling in the mechanism of Ca2+ influx in endothelial cells via Trp channels (ii) study the role of caveolin-1 in the mechanism of Ca2+ influx through Trp channels and its role in the mechanism of increased endothelial permeability, and (iii) investigate the effects of inflammatory mediators, TNFa and lipopolysaccharide, on the functional expression of Trp channels and its role in the mechanism of increase in endothelial permeability. The proposed studies will utilize cell and in vivo approaches involving Src null (Src -/-), caveolin-1 null (cav1 -/-), and Trp4 null (Trp4 -/-) mice to address the role of Trp channels in activating Ca2+ influx in endothelial cells and in the mechanism of increased endothelial permeability. With the achievement of these studies, we will be able to provide new molecular insight into understanding the mechanism of vascular injury and tissue inflammation associated with states of hypercoagulation such as with sepsis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058531-06
Application #
6699675
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Somers, Scott D
Project Start
1999-02-01
Project End
2007-01-31
Budget Start
2004-02-01
Budget End
2005-01-31
Support Year
6
Fiscal Year
2004
Total Cost
$280,566
Indirect Cost
Name
University of Illinois at Chicago
Department
Pharmacology
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Sundivakkam, Premanand C; Natarajan, Viswanathan; Malik, Asrar B et al. (2013) Store-operated Ca2+ entry (SOCE) induced by protease-activated receptor-1 mediates STIM1 protein phosphorylation to inhibit SOCE in endothelial cells through AMP-activated protein kinase and p38? mitogen-activated protein kinase. J Biol Chem 288:17030-41
Sundivakkam, Premanand C; Freichel, Marc; Singh, Vandana et al. (2012) The Ca(2+) sensor stromal interaction molecule 1 (STIM1) is necessary and sufficient for the store-operated Ca(2+) entry function of transient receptor potential canonical (TRPC) 1 and 4 channels in endothelial cells. Mol Pharmacol 81:510-26
Thippegowda, Prabhakar B; Singh, Vandana; Sundivakkam, Premanand C et al. (2010) Ca2+ influx via TRPC channels induces NF-kappaB-dependent A20 expression to prevent thrombin-induced apoptosis in endothelial cells. Am J Physiol Cell Physiol 298:C656-64
Minshall, Richard D; Vandenbroucke, Emily E; Holinstat, Michael et al. (2010) Role of protein kinase Czeta in thrombin-induced RhoA activation and inter-endothelial gap formation of human dermal microvessel endothelial cell monolayers. Microvasc Res 80:240-9
Bair, Angela M; Thippegowda, Prabhakar B; Freichel, Marc et al. (2009) Ca2+ entry via TRPC channels is necessary for thrombin-induced NF-kappaB activation in endothelial cells through AMP-activated protein kinase and protein kinase Cdelta. J Biol Chem 284:563-74
Sundivakkam, Premanand C; Kwiatek, Angela M; Sharma, Tiffany T et al. (2009) Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells. Am J Physiol Cell Physiol 296:C403-13
Xue, Jiaping; Thippegowda, Prabhakar B; Hu, Guochang et al. (2009) NF-kappaB regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-kappaB site in the ICAM-1 gene. Physiol Genomics 38:42-53
Tiruppathi, Chinnaswamy; Shimizu, Jun; Miyawaki-Shimizu, Kayo et al. (2008) Role of NF-kappaB-dependent caveolin-1 expression in the mechanism of increased endothelial permeability induced by lipopolysaccharide. J Biol Chem 283:4210-8
Kwiatek, Angela M; Minshall, Richard D; Cool, David R et al. (2006) Caveolin-1 regulates store-operated Ca2+ influx by binding of its scaffolding domain to transient receptor potential channel-1 in endothelial cells. Mol Pharmacol 70:1174-83
Tiruppathi, Chinnaswamy; Ahmmed, Gias U; Vogel, Stephen M et al. (2006) Ca2+ signaling, TRP channels, and endothelial permeability. Microcirculation 13:693-708

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