Numerous diseases affecting the GI tract, ranging from secretory diarrhea to cystic fibrosis, are characterized by dysregulation of epithelial C1- secretion. This project originally identified that ammonium ion (NH4+, normally present at high concentrations in the colonic lumen) may be a novel endogenous regulator of C1- secretion via effects via effects on K+ channels and begins to define the interaction of NH4+ with the basolateral membrane K+ transporters also required for Cl-secretion. Based on work already accomplished, the current application considers how altered K+ channel regulation may influence various intestinal disease states. Preliminary data indicate that the ammonia-derived oxidant monochloramine (NH2Cl) may contribute to the diarrhea of colitis by potentiating Ca2+- dependent K+ channels. Experiments also suggest that docosahexaenoic acid (DHA, a component of fish oil) can augment Ca+2- dependent K+ channels, finding of particular interest as DHA begins clinical evaluation as therapy in CF. Preliminary findings suggest that the actin cytoskeleton an functionally alter Ca2+-dependent K+ channels, and conversely, that these K+ channels can modulate cell functions such as epithelial restitution that involve actin remodeling. Three sets of studies are proposed. First, the impact of ammonia on colonic epithelial transport will be further characterized in cultured epithelial ells and in human colonic mucosal preparations, with attention to the interaction of NH4+ with the basolateral Na+-k+-2Cl- co- transporter, Na+_K+ ATPase, and K+ channels. Second, potentiation of basolateral Ca+2-dependent K+ channels by cAMP and NH2Cl will be explored using cultured epithelial cells as model systems with the goal of defining a common mechanism for K+ channel potentiation by these seemingly diverse stimuli. The potential for therapeutic modulation of basolateral K + channels will be explored, specifically examining wheth4r docosahexaenoic acid (DHA) can augment Ca2+-dependent Cl- secretion in T84 cells and human colon, and, if so, to determine its mechanism of action. Finally, the studies will define the effect of chemical manipulation of F-actin on Ca2+-dependent K+ channel regulation and extend preliminary findings suggesting that K+ channel regulation affects the actin-regulated process of epithelial restitution. These studies highlight the importance of basolateral K+ channels in the regulation of secretion and other epithelial functions and reinforce their potential as targets for new drug design.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK051630-05
Application #
6196979
Study Section
Surgery and Bioengineering Study Section (SB)
Program Officer
May, Michael K
Project Start
1996-09-01
Project End
2001-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
5
Fiscal Year
2000
Total Cost
$348,000
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02215
Worrell, Roger T; Merk, Lisa; Matthews, Jeffrey B (2008) Ammonium transport in the colonic crypt cell line, T84: role for Rhesus glycoproteins and NKCC1. Am J Physiol Gastrointest Liver Physiol 294:G429-40
Del Castillo, Isabel Calvo; Fedor-Chaiken, Mary; Song, J Cecilia et al. (2005) Dynamic regulation of Na(+)-K(+)-2Cl(-) cotransporter surface expression by PKC-{epsilon} in Cl(-)--secretory epithelia. Am J Physiol Cell Physiol 289:C1332-42
Li, Hong C; Szigligeti, Peter; Worrell, Roger T et al. (2005) Missense mutations in Na+:HCO3- cotransporter NBC1 show abnormal trafficking in polarized kidney cells: a basis of proximal renal tubular acidosis. Am J Physiol Renal Physiol 289:F61-71
Worrell, Roger T; Best, Alison; Crawford, Oscar R et al. (2005) Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent. Am J Physiol Gastrointest Liver Physiol 289:G768-78
Li, Hong C; Worrell, Roger T; Matthews, Jeffrey B et al. (2004) Identification of a carboxyl-terminal motif essential for the targeting of Na+-HCO-3 cotransporter NBC1 to the basolateral membrane. J Biol Chem 279:43190-7
Lotz, Margaret M; Wang, Helen; Song, Jaekyung Cecilia et al. (2004) K+ channel inhibition accelerates intestinal epithelial cell wound healing. Wound Repair Regen 12:565-74
Silva, Milton; Song, Cecilia; Nadeau, William J et al. (2004) Salmonella typhimurium SipA-induced neutrophil transepithelial migration: involvement of a PKC-alpha-dependent signal transduction pathway. Am J Physiol Gastrointest Liver Physiol 286:G1024-31
Worrell, Roger T; Oghene, Jennifer; Matthews, Jeffrey B (2004) Ammonium effects on colonic Cl- secretion: anomalous mole fraction behavior. Am J Physiol Gastrointest Liver Physiol 286:G14-22
Mayol, Julio M; Alarma-Estrany, Pilar; O'Brien, Timothy C et al. (2003) Electrogenic ion transport in mammalian colon involves an ammonia-sensitive apical membrane K+ conductance. Dig Dis Sci 48:116-25
Yoo, James; Nichols, Anthony; Song, Jaekyung C et al. (2003) Bryostatin-1 attenuates TNF-induced epithelial barrier dysfunction: role of novel PKC isozymes. Am J Physiol Gastrointest Liver Physiol 284:G703-12

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