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Am J Physiol. 1999 Aug;277(2):G292-305. doi: 10.1152/ajpgi.1999.277.2.G292.

Development of an endogenous epithelial Na(+)/H(+) exchanger (NHE3) in three clones of caco-2 cells.

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1
Departments of Medicine and Physiology, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Abstract

Expression of endogenous Na(+)/H(+) exchangers (NHEs) NHE3 and NHE1 at the apical (AP) and basolateral (BL) membrane domains was investigated in three clones (ATCC, PF-11, and TC-7) derived from the human adenocarcinoma cell line Caco-2. In all three clones, NHE1 was the only isoform detected at the BL domain during 3 to 22 postconfluent days (PCD). In clone PF-11, the BL NHE1 activity increased up to 7 PCD and remained stable thereafter. Both NHE1 and NHE3 were found at the AP domain at 3 PCD and contributed 67 and 33% to the total AP Na(+)/H(+) exchange, respectively. The AP NHE3 activity increased significantly from 3 to 22 PCD, from 93 to 450 microM H(+)/s, whereas AP NHE1 activity decreased from 192 to 18 microM H(+)/s during that time. Similar results were obtained with the ATCC clone, whereas very little AP NHE3 activity was observed in clone TC-7. Surface biotinylation and indirect immunofluorescence confirmed these results and also suggested an increase in the number of cells expressing NHE3 being the major mechanism of the observed overall increase in NHE3 activity in PF-11 and ATCC clones. Phorbol 12-myristate 13-acetate (PMA, 1 microM) acutely inhibited NHE3 activity by 28% of control, whereas epidermal growth factor (EGF, 200 ng/ml) stimulated the activity by 18%. The effect of PMA was abolished by the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, suggesting involvement of PKC in the PMA-induced inhibition of NHE3. Similar magnitude of inhibition by PMA and stimulation by EGF was observed at 7 and 17 PCD, suggesting the development of regulatory mechanisms in the early postconfluent period. Taken together, these data suggest a close similarity of membrane targeting and regulation of endogenous NHE3 between Caco-2 cells and native small intestinal epithelial cells and support the usefulness of some Caco-2 cell clones as an in vitro model for studies on physiology of NHE3 in the intestinal epithelium.

PMID:
10444443
DOI:
10.1152/ajpgi.1999.277.2.G292
[Indexed for MEDLINE]

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