Document Detail

Carbachol regulation of rabbit ileal brush border Na+-H+ exchanger 3 (NHE3) occurs through changes in NHE3 trafficking and complex formation and is Src dependent.
MedLine Citation:
PMID:  14978207     Owner:  NLM     Status:  MEDLINE    
The epithelial brush border membrane (BBM) Na(+)-H(+) exchanger 3 (NHE3) is the major transport protein responsible for ileal electroneutral Na(+) absorption. We have previously shown that ileal BBM NHE3 activity is rapidly inhibited by carbachol, an agonist that mimics cholinergic activation in digestion. In this study, we investigated the mechanisms involved in this NHE3 inhibition. Carbachol decreased the amount of ileal Na(+) absorptive cell BBM NHE3 within 10 min of exposure. Based on OptiPrep gradient centrifugation, carbachol increased the amount of NHE3 in early endosomes and decreased the amount of NHE3 in BBM, consistent with effects on NHE3 trafficking. The decrease in BBM NHE3 occurred in the detergent-soluble BBM fraction with no change in the amount of NHE3 in the BBM detergent-resistant membranes. The size of BBM NHE3 complexes increased in carbachol-exposed ileum, as studied with sucrose gradient centrifugation. The NHE3 complex size increased in the total BBM, but did not change in the detergent-soluble fraction. This suggests that carbachol treatment enhanced the association of proteins with NHE3 complexes specifically in the detergent-resistant fraction of ileal BBM. NHERF2, alpha-actinin-4 and protein kinase C were among those NHE3-associated proteins because they were more efficiently coimmunoprecipitated from total BBM after carbachol treatment. Moreover, Src was involved in the carbachol-mediated inhibition since: (1) c-Src was rapidly activated in the detergent-resistant membranes by carbachol; and (2) carbachol inhibition of ileal Na(+) absorption was completely abolished by the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, the carbachol-induced increase in the size of NHE3-containing complexes was reversed by PP2. These data demonstrate that regulation of NHE3 activity by carbachol can be achieved at several interrelated levels: (1) the subcellular level, at which NHE3 is rapidly endocytosed from BBM to endocytic vesicles upon treatment with carbachol; (2) multiple BBM pools, in which carbachol selectively decreases the amount of NHE3 in the BBM detergent-soluble fraction but not the detergent-resistant membrane; and (3) the molecular level, at which NHE3 complex-associated proteins can be changed upon carbachol treatment, with carbachol leading to larger BBM NHE3 complexes and increased co-IP of NHERF2 with alpha-actinin-4 and activated PKC. The study further describes NHE3 presence simultaneously in multiple dynamic BBM pools in which NHE3 distribution and associated proteins are altered as part of carbachol-induced and Src-mediated rapid signal transduction, which decreases the amount of BBM NHE3 and thus inhibits NHE3 activity.
Xuhang Li; Huiping Zhang; Alice Cheong; Sharon Leu; Yueping Chen; Christian G Elowsky; Mark Donowitz
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.     Date:  2004-02-20
Journal Detail:
Title:  The Journal of physiology     Volume:  556     ISSN:  0022-3751     ISO Abbreviation:  J. Physiol. (Lond.)     Publication Date:  2004 May 
Date Detail:
Created Date:  2004-04-30     Completed Date:  2005-03-21     Revised Date:  2013-06-09    
Medline Journal Info:
Nlm Unique ID:  0266262     Medline TA:  J Physiol     Country:  England    
Other Details:
Languages:  eng     Pagination:  791-804     Citation Subset:  IM    
Department of Medicine, GI Division, John Hopkins University School of Medicine, Baltimore, MD 21205-2195, USA .
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MeSH Terms
Actinin / metabolism*
Biological Transport / drug effects
Blotting, Western
Carbachol / pharmacology*
Endosomes / chemistry
Epithelial Cells / cytology,  drug effects,  metabolism
Ileum / drug effects,  metabolism,  physiology*
Microscopy, Fluorescence
Microvilli / drug effects,  metabolism
Protein Binding / drug effects
Protein Transport / drug effects
Protein-Tyrosine Kinases / analysis,  metabolism
Pyrimidines / pharmacology
Sodium-Hydrogen Antiporter / metabolism,  physiology*
Water / analysis,  metabolism
src-Family Kinases / antagonists & inhibitors,  metabolism,  physiology*
Grant Support
Reg. No./Substance:
0/AG 1879; 0/Pyrimidines; 0/Sodium-Hydrogen Antiporter; 0/sodium-hydrogen exchanger 3; 11003-00-2/Actinin; 51-83-2/Carbachol; 7732-18-5/Water; EC Kinases; EC tyrosine-protein kinase; EC Kinases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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