Ammonia exerts profound and diverse biological effects on most mammalian cells. The normal colon is exposed to unusually high ammonia concentrations, yet the impact of this simple and ubiquitous substance is largely unknown. The broad intent of this proposal is to explore the interaction of ammonia with the events that comprise regulated intestinal CI- secretion using the T84 human intestinal epithelial cell line and native mammalian tissue as model systems. This proposal builds upon preliminary data that suggest ammonia is a previously unsuspected regulator of colonic electrolyte transport, a finding that may have substantial implications for intestinal function during health and disease. Two groups of studies are planned that will use electrophysiologic methods and isotopic flux analysis, fluorescence-based optical techniques, and cell surface labeling techniques. The first set of studies will expand upon preliminary characterization of the inhibitory effects of ammonia on regulated CI- secretion, comparing results in a cultured cell line to those in a native preparation and specifically examining the apparent selectivity of ammonia for cyclic nucleotide dependent agonists. The basis for the asymmetry in inhibitory potency of ammonia depending upon route of exposure will be explored. The second set of studies will attempt to define the relevant target of ammonia in epithelial cells that accounts for its inhibitory action. These experiments utilize ammonia and related weak bases as physiologic probes into the cellular mechanisms underlying this fundamental and widely distributed epithelial transport process. The hypothesis that ammonia reduces epithelial secretory capacity by perturbing the membrane cycling events that control cell surface expression of specific transport proteins will be addressed. The proposed studies will lay the groundwork for future formalized studies addressing the potential role of ammonia as a pathogenic cofactor in intestinal disease. Because several weak bases appear to share with ammonia the ability to inhibit CI- secretion, these investigations may form the basis for the development of new pharmacologic approaches to the treatment of secretory diarrheal illnesses. By extension, these experiments may yield considerable insight into the biological actions of amines and resolve some of the controversy surrounding their effects on membrane traffic events. These studies may also have broad implications for regulated transport events in organ systems exposed to high levels of ammonia under normal circumstances, during infection with ureolytic organisms, or during hyperammonemic states.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Surgery and Bioengineering Study Section (SB)
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Beth Israel Deaconess Medical Center
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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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