The long term GOAL of this proposal is to identify and study the regulation of ileal Na+/H+ exchangers. Rabbit ileal villus cells have two forms of Na+/H+ exchangers which are distinct in terms of function (transcellular Na absorption vs ph/volume regulation), pharmacology (amiloride sensitivity) and localization (apical vs basolateral). The apical membrane Na+/H+ exchanger is part of neutral NaCl absorption which is inhibited in diarrheal diseases. We have isolated and functionally expressed in fibroblasts deficient in Na+/H+ exchange, a cDNA encoding a rabbit ileal villus basolateral membrane Na*/H* exchanger (NHE) and cloned a partial cDNA encoding a Na+/H+ exchanger related protein (NHERP) which may be the apical membrane Na+/H+ exchanger. Therefore, we propose to obtain a full-length cDNA encoding the NHERP; analyze the tissue distribution of NHERP; and use genomic Southern blot analysis to further confirm that NHE and NHERP are encoded by two separate genes. We will confirm that NHE and NHERP function as Na+/H+ exchangers by transient and stable transfection of the cDNAs into the Na+/H+ exchange deficient fibroblasts; Na+/H+ exchange activity will be determined by demonstration of Na+-dependent recovery from an acid load monitored with fluoresence by the intracellular ph-sensitive dye, BCECF. In contrast to ileal villus cells, crypt cells do not have apical membrane Na+/H+ exchangers. In order to correlate the physiological roles of NHE and NHERP in the ileal epithelium, we will localize the NHE and NHERP messages in cells along the villuscrypt axis by Northern blotting and in situ hybridization using NHE and NHERP cDNAs as probes and localize NHE and NHERP by Western blotting and immunocytochemistry using antibodies raised against NHE and NHERP. The apical membrane Na+/H+ exchanger is regulated by protein kinases. We will establish the functional role of the phosphorylation of NHERP in regulation of Na+/H+ exchange in ileal Na+ absorbing cells by demonstrating that the NHERP is a phosphoprotein and studying the effects of second messenger agonists on its phosphorylation by in vitro and in vivo phosphorylation combined with Western blotting. To provide the basis for identification of the phosphorylation site(s) of the Na+/H+ exchanger, we will identify the phosphoamino acids in NHERP which form in response to elevation in C kinase and A kinase. Then, we will study the structure-funtion relationship of NHE and NHERP by confirming that NHE and NHERP are glycoproteins and identifying ion transporting domains and domains involved in protein kinase regulation of NHE and NHERP by deletion/expression analysis.

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 #
5R29DK043778-04
Application #
2143280
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1991-06-15
Project End
1996-05-31
Budget Start
1994-06-01
Budget End
1995-05-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Nath, S K; Kambadur, R; Yun, C H et al. (1999) NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation. Am J Physiol 276:C873-82
Janecki, A J; Montrose, M H; Tse, C M et al. (1999) Development of an endogenous epithelial Na(+)/H(+) exchanger (NHE3) in three clones of caco-2 cells. Am J Physiol 277:G292-305
Wormmeester, L; Sanchez de Medina, F; Kokke, F et al. (1998) Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange. Am J Physiol 274:C1261-72
Janecki, A J; Montrose, M H; Zimniak, P et al. (1998) Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J Biol Chem 273:8790-8
Yip, J W; Ko, W H; Viberti, G et al. (1997) Regulation of the epithelial brush border Na+/H+ exchanger isoform 3 stably expressed in fibroblasts by fibroblast growth factor and phorbol esters is not through changes in phosphorylation of the exchanger. J Biol Chem 272:18473-80
Hoogerwerf, W A; Tsao, S C; Devuyst, O et al. (1996) NHE2 and NHE3 are human and rabbit intestinal brush-border proteins. Am J Physiol 270:G29-41
Nath, S K; Hang, C Y; Levine, S A et al. (1996) Hyperosmolarity inhibits the Na+/H+ exchanger isoforms NHE2 and NHE3: an effect opposite to that on NHE1. Am J Physiol 270:G431-41
Yun, C H; Tse, C M; Donowitz, M (1995) Chimeric Na+/H+ exchangers: an epithelial membrane-bound N-terminal domain requires an epithelial cytoplasmic C-terminal domain for regulation by protein kinases. Proc Natl Acad Sci U S A 92:10723-7
Yun, C H; Tse, C M; Nath, S K et al. (1995) Mammalian Na+/H+ exchanger gene family: structure and function studies. Am J Physiol 269:G1-11
Brant, S R; Yun, C H; Donowitz, M et al. (1995) Cloning, tissue distribution, and functional analysis of the human Na+/N+ exchanger isoform, NHE3. Am J Physiol 269:C198-206

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