Bacterial infections of the gastrointestinal (Gl) tract cause 300,000 hospitalizations and 5,000 deaths each year in the United States. We previously demonstrated that the galanin-1 receptor (Gal1R) is up-regulated in colonic epithelial cells in response to infection with diverse bacterial pathogens. When present, Gal1R activation by ligand normally present in enteric nerves results in CI- secretion, a mechanism that is responsible for a large component of the excess ntestinal fluid secretion observed in infectious diarrhea. We herein show that the increase in bacterial pathogen-induced colonic fluid secretion mediated by the GaUR occurs via a cAMP-independent but Ca2+-dependent pathway that is inhibitable by pertussis toxin, suggesting that this receptor couples to a member of the Galphi(i) family of heterotrimeric G proteins. We also provide evidence indicating that the Gal1R increases colonic CI- secretion via a calcium-activated CI- channel (CLCA). Thus we extend our novel and mechanistic hypothesis that Gal1-R up-regulation accounts for a significant component of the excessive fluid secretion observed in infectious diarrhea and propose that this occurs via a Ga ph ( )/CLCA-dependent pathway. To evaluate this hypothesis we will: 1. Determine how the Gal1R activates its cognate G protein(s) to cause Cl- secretion. The specific G protein(s) coupling to the Gal1R along with the receptor sites involved in this coupling will be identified. We will use the nonhydrolyzable GTP photoaffinity analog azidoanilido GTP to identify the G protein(s) physically coupling to the Gal1R. We will use the novel method of synthetic peptides on membrane support (SPOT) to obtain initial information as to where the G protein subunit(s) bind to the Gal1R. These data will be modeled using GRASP, AutoDock and DOCK to obtain initial information as to sites of Gal1R- Galphi(i) binding. These sites will be tested by introducing mini-peptides into permeabilized cells that are predicted to block Gal1R- Galphi(i) binding;those abrogating galanin-induced signaling will confirm the regions so identified as critical to this interaction. We will then: 2. Identify the Gall R-activated CI- channel mediating galanin-induced CI- secretion. Although our preliminary data strongly suggests that galanin increases colonic fluid secretion in a CFTR-independent manner by activating a CLCA chloride channel, increased amounts of colonic fluid can also result from activation of CLC chloride channels, K+ channels, or decreased Na+ absorption. To do this we will study enteric pathogen infection in wild type mice, and in mice genetically incapable of expressing Gal1R (Gal1R-/-) and in mice genetically incapable of expressing CFTR (CFTR-/-). Studying the Gal1R-/- mice will allow us to determine the contribution of galanin-mediated colonic fluid secretion, and studying the CFTR-/- will allow us to determine the direct contributions of CFTR-independent pathways. Overall these experiments will allow us to elucidate the mechanism whereby galanin causes CI- secretion, and generate information useful for the generation of agents useful in the treatment of diarrhea due to enteric pathogen infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Program Projects (P01)
Project #
5P01DK067887-04
Application #
7905696
Study Section
Special Emphasis Panel (ZDK1)
Project Start
Project End
Budget Start
2009-08-01
Budget End
2010-07-31
Support Year
4
Fiscal Year
2009
Total Cost
$193,061
Indirect Cost
Name
University of Illinois at Chicago
Department
Type
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
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