Induction of Na+/K+/2Cl- cotransporter expression mediates chronic potentiation of intestinal epithelial Cl- secretion by EGF

Fiona O'Mahony, Ferial Toumi, Magdalena S Mroz, Gail Ferguson, Stephen J Keely

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl(-) secretion was measured as changes in short-circuit current (Isc) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced Isc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na+/K+/2Cl(-) cotransporter (NKCC1) inhibitor bumetanide (100 microM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl(-) secretion. Neither basal nor agonist-stimulated levels of intracellular Ca2+ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl(-) secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 +/- 21.9% of that in control cells (n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.

Original languageEnglish
Pages (from-to)C1362-70
JournalAmerican Journal of Physiology - Cell Physiology
Volume294
Issue number6
DOIs
Publication statusPublished - Jun 2008

Fingerprint

Epidermal Growth Factor
Epidermal Growth Factor Receptor
Member 2 Solute Carrier Family 12
Bumetanide
Dactinomycin
Second Messenger Systems
Electrolytes
Intercellular Signaling Peptides and Proteins
Up-Regulation
Polymerase Chain Reaction
Messenger RNA

Keywords

  • Bumetanide/pharmacology
  • Calcium/metabolism
  • Cell Line
  • Chlorides/metabolism
  • Cholecystokinin/metabolism
  • Cyclic AMP-Dependent Protein Kinases/metabolism
  • Dactinomycin/pharmacology
  • Epidermal Growth Factor/metabolism
  • ErbB Receptors/metabolism
  • Humans
  • Intestinal Mucosa/drug effects
  • Membrane Potentials
  • Neuregulin-1/metabolism
  • RNA, Messenger/metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sodium Potassium Chloride Symporter Inhibitors/pharmacology
  • Sodium-Potassium-Chloride Symporters/genetics
  • Solute Carrier Family 12, Member 2
  • Time Factors
  • Transforming Growth Factor alpha/metabolism
  • Up-Regulation

Cite this

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title = "Induction of Na+/K+/2Cl- cotransporter expression mediates chronic potentiation of intestinal epithelial Cl- secretion by EGF",
abstract = "Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl(-) secretion was measured as changes in short-circuit current (Isc) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced Isc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na+/K+/2Cl(-) cotransporter (NKCC1) inhibitor bumetanide (100 microM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl(-) secretion. Neither basal nor agonist-stimulated levels of intracellular Ca2+ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl(-) secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 +/- 21.9{\%} of that in control cells (n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.",
keywords = "Bumetanide/pharmacology, Calcium/metabolism, Cell Line, Chlorides/metabolism, Cholecystokinin/metabolism, Cyclic AMP-Dependent Protein Kinases/metabolism, Dactinomycin/pharmacology, Epidermal Growth Factor/metabolism, ErbB Receptors/metabolism, Humans, Intestinal Mucosa/drug effects, Membrane Potentials, Neuregulin-1/metabolism, RNA, Messenger/metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sodium Potassium Chloride Symporter Inhibitors/pharmacology, Sodium-Potassium-Chloride Symporters/genetics, Solute Carrier Family 12, Member 2, Time Factors, Transforming Growth Factor alpha/metabolism, Up-Regulation",
author = "Fiona O'Mahony and Ferial Toumi and Mroz, {Magdalena S} and Gail Ferguson and Keely, {Stephen J}",
year = "2008",
month = "6",
doi = "10.1152/ajpcell.00256.2007",
language = "English",
volume = "294",
pages = "C1362--70",
journal = "American Journal of Physiology - Cell Physiology",
issn = "0363-6143",
publisher = "American Physiological Society",
number = "6",

}

Induction of Na+/K+/2Cl- cotransporter expression mediates chronic potentiation of intestinal epithelial Cl- secretion by EGF. / O'Mahony, Fiona; Toumi, Ferial; Mroz, Magdalena S; Ferguson, Gail; Keely, Stephen J.

In: American Journal of Physiology - Cell Physiology, Vol. 294, No. 6, 06.2008, p. C1362-70.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Induction of Na+/K+/2Cl- cotransporter expression mediates chronic potentiation of intestinal epithelial Cl- secretion by EGF

AU - O'Mahony, Fiona

AU - Toumi, Ferial

AU - Mroz, Magdalena S

AU - Ferguson, Gail

AU - Keely, Stephen J

PY - 2008/6

Y1 - 2008/6

N2 - Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl(-) secretion was measured as changes in short-circuit current (Isc) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced Isc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na+/K+/2Cl(-) cotransporter (NKCC1) inhibitor bumetanide (100 microM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl(-) secretion. Neither basal nor agonist-stimulated levels of intracellular Ca2+ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl(-) secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 +/- 21.9% of that in control cells (n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.

AB - Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl(-) secretion was measured as changes in short-circuit current (Isc) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced Isc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na+/K+/2Cl(-) cotransporter (NKCC1) inhibitor bumetanide (100 microM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl(-) secretion. Neither basal nor agonist-stimulated levels of intracellular Ca2+ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl(-) secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 +/- 21.9% of that in control cells (n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.

KW - Bumetanide/pharmacology

KW - Calcium/metabolism

KW - Cell Line

KW - Chlorides/metabolism

KW - Cholecystokinin/metabolism

KW - Cyclic AMP-Dependent Protein Kinases/metabolism

KW - Dactinomycin/pharmacology

KW - Epidermal Growth Factor/metabolism

KW - ErbB Receptors/metabolism

KW - Humans

KW - Intestinal Mucosa/drug effects

KW - Membrane Potentials

KW - Neuregulin-1/metabolism

KW - RNA, Messenger/metabolism

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Sodium Potassium Chloride Symporter Inhibitors/pharmacology

KW - Sodium-Potassium-Chloride Symporters/genetics

KW - Solute Carrier Family 12, Member 2

KW - Time Factors

KW - Transforming Growth Factor alpha/metabolism

KW - Up-Regulation

U2 - 10.1152/ajpcell.00256.2007

DO - 10.1152/ajpcell.00256.2007

M3 - Article

C2 - 18400987

VL - 294

SP - C1362-70

JO - American Journal of Physiology - Cell Physiology

JF - American Journal of Physiology - Cell Physiology

SN - 0363-6143

IS - 6

ER -