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.?
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