In the intestine, electrogenic Cl-secretion forms the basis for a variety of pathologic states characterized by secretory diarrhea, including antibiotic-associated colitis, bacterial toxin-mediated diarrhea, ostomy diarrhea, certain neuroendocrine syndromes, and numerous inflammatory diseases of the intestine. Current models of electrogenic Cl secretion emphasize regulation of electrogenic Cl secretion at the level of apical membrane Cl channels because of the recently recognized relationship of this pathway to the pathogenesis of cystic fibrosis. In contrast, little is known about the regulation of basolateral membrane transporter function. In order to maintain cellular composition during high rates of secretion, basolateral Cl entry and apical Cl exit must be precisely matched, yet the mechanism by which coordination of apical and basolateral transport events (""""""""crosstalk"""""""") is accomplished is incompletely understood. The general goal of this proposal is to examine at a fundamental level the regulation of the Na+/K+/2Cl cotransporter, the basolateral Cl uptake mechanism of Cl secreting epithelial cells. The Na+/K+/2Cl cotransporter is a highly conserved membrane transporter which, in addition to its integral role in salt and water transport in both secretory and absorptive epithelia, may be involved in cell volume regulation in both epithelial and non-epithelial cells. Accumulating evidence suggests that the regulation of the Na+/K+/2Cl cotransporter is a complex process involving protein phosphorylation and alteration in numbers of transporters in the plasmalemma. Preliminary data for this project suggest the novel possibility that the F-actin cytoskeleton may dynamically influence these events. The studies proposed will be carried out using established cultured human intestinal cell lines as model systems. The specific goals of this project are 1) to characterize functionally the regulation of the basolateral membrane Na+/K/2Cl cotransporter in response to cAMP-mediated agonist, non-cAMP secretagogue, and osmotic perturbation, 2) to correlate regulated changes in cotransporter function with changes in the number of active cotransporter sites on the basolateral membrane, 3) to define the possible role of the F-actin microfilament system in regulation of cotransporter number and activity, and, 4) to identify some of the intracellular signalling pathways involved in modulation of cotransporter function. The propose studies should improve our understanding of electrogenic Cl secretion by elucidating the mechanism for regulation of Na+/K+/2Cl cotransport in intestinal epithelial cells. By determining the interaction between an ion-transporting integral membrane protein (the cotransporter) and the F-actin microfilament system, these experiments may prove to have broader implications for other important ion transport processes and should yield insight into the function of the cytoskeleton in epithelial cells. It is hoped that an understanding of fundamental mechanisms underlying electrogenic Cl secretion will establish a rational basis for designing new therapeutic approaches to the problem of secretory diarrhea.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29DK048010-01
Application #
2148015
Study Section
Surgery and Bioengineering Study Section (SB)
Project Start
1994-05-01
Project End
1999-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02215
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Tang, Jun; Bouyer, Patrice; Mykoniatis, Andreas et al. (2010) Activated PKC{delta} and PKC{epsilon} inhibit epithelial chloride secretion response to cAMP via inducing internalization of the Na+-K+-2Cl- cotransporter NKCC1. J Biol Chem 285:34072-85
Mykoniatis, Andreas; Shen, Le; Fedor-Chaiken, Mary et al. (2010) Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway. Am J Physiol Cell Physiol 298:C85-97
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Tally, K J; Hrnjez, B J; Smith, J A et al. (1996) Adenosine scavenging: a novel mechanism of chloride secretory control in intestinal epithelial cells. Surgery 120:248-54
Matthews, J B; Tally, K J; Smith, J A et al. (1995) Activation of Cl secretion during chemical hypoxia by endogenous release of adenosine in intestinal epithelial monolayers. J Clin Invest 96:117-25
Matthews, J B; Tally, K J; Smith, J A (1995) Activation of intestinal Na-K-2Cl cotransport by 5'-AMP requires F-actin remodeling. Am J Surg 169:50-5;discussion 55-6
Matthews, J B; Smith, J A; Nguyen, H (1995) Modulation of intestinal chloride secretion at basolateral transport sites: opposing effects of cyclic adenosine monophosphate and phorbol ester. Surgery 118:147-52;discussion 152-3

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