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

Andrzej J. Janecki, Marshall H. Montrose, Chung Ming Tse, Fermin Sanchez De Medina, Alain Zweibaum, Mark Donowitz

Research output: Contribution to journalArticle

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 μM H+/s, whereas AP NHE1 activity decreased from 192 to 18 μM 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 μM) 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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume277
Issue number2 40-2
StatePublished - 1999

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Sodium-Hydrogen Antiporter
Caco-2 Cells
Clone Cells
Epidermal Growth Factor
Protein Kinase C
Biotinylation
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
Membranes
Protein C Inhibitor
Intestinal Mucosa
Protein Kinase Inhibitors
Indirect Fluorescent Antibody Technique
Protein Isoforms
Adenocarcinoma
Acetates
Cell Count
Epithelial Cells
Cell Line
pseudoginsenoside F11

Keywords

  • Epidermal growth factor
  • Phorbol ester
  • Protein kinase C

ASJC Scopus subject areas

  • Gastroenterology
  • Physiology
  • Physiology (medical)

Cite this

Development of an endogenous epithelial Na+/H+ exchanger (NHE3) in three clones of Caco-2 cells. / Janecki, Andrzej J.; Montrose, Marshall H.; Tse, Chung Ming; De Medina, Fermin Sanchez; Zweibaum, Alain; Donowitz, Mark.

In: American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 277, No. 2 40-2, 1999.

Research output: Contribution to journalArticle

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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 μM H+/s, whereas AP NHE1 activity decreased from 192 to 18 μM 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 μM) 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.",
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T1 - Development of an endogenous epithelial Na+/H+ exchanger (NHE3) in three clones of Caco-2 cells

AU - Janecki, Andrzej J.

AU - Montrose, Marshall H.

AU - Tse, Chung Ming

AU - De Medina, Fermin Sanchez

AU - Zweibaum, Alain

AU - Donowitz, Mark

PY - 1999

Y1 - 1999

N2 - 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 μM H+/s, whereas AP NHE1 activity decreased from 192 to 18 μM 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 μM) 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.

AB - 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 μM H+/s, whereas AP NHE1 activity decreased from 192 to 18 μM 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 μM) 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.

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