Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers

Cynthia Louise Sears, F. Firoozmand, A. Mellander, F. G. Chambers, I. G. Eromar, A. G M Bot, B. Scholte, H. R. De Jonge, Mark Donowitz

Research output: Contribution to journalArticle

Abstract

The involvement of tyrosine phosphorylation in the regulation of epithelial cell Cl- secretion is unknown. Therefore, the purpose of these studies was to determine if tyrosine kinase activation was involved in the regulation of Cl- secretion, using the tyrosine kinase inhibitors, genistein and tyrphostin 47, and human intestinal epithelial cells (T84 cells) as an intestinal Cl- secretory model. Genistein rapidly but reversibly stimulated sustained apical Cl- secretion in monolayers of T84 cells without increasing intracellular cyclic nucleotides or Ca2+ levels. Tyrphostin 47 also stimulated Cl- secretion in T84 monolayers, although it was short-lived. Transfection experiments in 3T3 fibroblasts and IEC-6 intestinal cells utilizing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) showed that genistein and tyrphostin 47 stimulated 125I efflux only in CFTR-transfected cells and not in CFTR-negative cells. Thus genistein- and tyrphostin 47-stimulated Cl- secretion involved CFTR. Genistein also acted synergistically with the Ca2+- and protein kinase C-dependent acetylcholine analogue, carbachol, to stimulate Cl- secretion in T84 monolayers. However, the Cl- secretory response to saturating concentrations of the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin, or the guanosine 3',5'- cyclic monophosphate (cGMP) agonist, Escherichia coli heat-stable enterotoxin, was not further enhanced by genistein. Although the mechanism of activation of Cl- secretion is unclear, these data suggest that tyrosine kinase activity limits basal Cl- secretion in T84 cells and that inhibition of T84 cell tyrosine kinase(s) stimulates apical membrane Cl- secretion, most likely through activation of the CFTR-Cl- channel. Moreover, genistein does not itself act through cAMP or cGMP elevation but appears to share a common Cl- secretory pathway with cyclic nucleotide-dependent agonists, whereas it augments the secretory responses to a Ca2+- and protein kinase C-dependent agonist.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume269
Issue number6 32-6
StatePublished - 1995

Fingerprint

Cystic Fibrosis Transmembrane Conductance Regulator
Genistein
cystic fibrosis
genistein
secretion
Protein-Tyrosine Kinases
tyrosine
agonists
phosphotransferases (kinases)
cells
Cyclic Nucleotides
cyclic nucleotides
Cyclic AMP
Protein Kinase C
protein kinase C
cyclic AMP
calcium
Epithelial Cells
epithelial cells
Secretory Pathway

Keywords

  • chloride
  • cystic fibrosis transmembrane conductance regulator
  • diarrhea
  • epithelium
  • Escherichia coli heat-stable enterotoxin
  • guanylate cyclase
  • human intestinal epithelial cell
  • protein kinases
  • tyrosine kinases

ASJC Scopus subject areas

  • Gastroenterology
  • Physiology
  • Agricultural and Biological Sciences(all)

Cite this

Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers. / Sears, Cynthia Louise; Firoozmand, F.; Mellander, A.; Chambers, F. G.; Eromar, I. G.; Bot, A. G M; Scholte, B.; De Jonge, H. R.; Donowitz, Mark.

In: American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 269, No. 6 32-6, 1995.

Research output: Contribution to journalArticle

Sears, CL, Firoozmand, F, Mellander, A, Chambers, FG, Eromar, IG, Bot, AGM, Scholte, B, De Jonge, HR & Donowitz, M 1995, 'Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers', American Journal of Physiology - Gastrointestinal and Liver Physiology, vol. 269, no. 6 32-6.
Sears, Cynthia Louise ; Firoozmand, F. ; Mellander, A. ; Chambers, F. G. ; Eromar, I. G. ; Bot, A. G M ; Scholte, B. ; De Jonge, H. R. ; Donowitz, Mark. / Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers. In: American Journal of Physiology - Gastrointestinal and Liver Physiology. 1995 ; Vol. 269, No. 6 32-6.
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T1 - Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers

AU - Sears, Cynthia Louise

AU - Firoozmand, F.

AU - Mellander, A.

AU - Chambers, F. G.

AU - Eromar, I. G.

AU - Bot, A. G M

AU - Scholte, B.

AU - De Jonge, H. R.

AU - Donowitz, Mark

PY - 1995

Y1 - 1995

N2 - The involvement of tyrosine phosphorylation in the regulation of epithelial cell Cl- secretion is unknown. Therefore, the purpose of these studies was to determine if tyrosine kinase activation was involved in the regulation of Cl- secretion, using the tyrosine kinase inhibitors, genistein and tyrphostin 47, and human intestinal epithelial cells (T84 cells) as an intestinal Cl- secretory model. Genistein rapidly but reversibly stimulated sustained apical Cl- secretion in monolayers of T84 cells without increasing intracellular cyclic nucleotides or Ca2+ levels. Tyrphostin 47 also stimulated Cl- secretion in T84 monolayers, although it was short-lived. Transfection experiments in 3T3 fibroblasts and IEC-6 intestinal cells utilizing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) showed that genistein and tyrphostin 47 stimulated 125I efflux only in CFTR-transfected cells and not in CFTR-negative cells. Thus genistein- and tyrphostin 47-stimulated Cl- secretion involved CFTR. Genistein also acted synergistically with the Ca2+- and protein kinase C-dependent acetylcholine analogue, carbachol, to stimulate Cl- secretion in T84 monolayers. However, the Cl- secretory response to saturating concentrations of the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin, or the guanosine 3',5'- cyclic monophosphate (cGMP) agonist, Escherichia coli heat-stable enterotoxin, was not further enhanced by genistein. Although the mechanism of activation of Cl- secretion is unclear, these data suggest that tyrosine kinase activity limits basal Cl- secretion in T84 cells and that inhibition of T84 cell tyrosine kinase(s) stimulates apical membrane Cl- secretion, most likely through activation of the CFTR-Cl- channel. Moreover, genistein does not itself act through cAMP or cGMP elevation but appears to share a common Cl- secretory pathway with cyclic nucleotide-dependent agonists, whereas it augments the secretory responses to a Ca2+- and protein kinase C-dependent agonist.

AB - The involvement of tyrosine phosphorylation in the regulation of epithelial cell Cl- secretion is unknown. Therefore, the purpose of these studies was to determine if tyrosine kinase activation was involved in the regulation of Cl- secretion, using the tyrosine kinase inhibitors, genistein and tyrphostin 47, and human intestinal epithelial cells (T84 cells) as an intestinal Cl- secretory model. Genistein rapidly but reversibly stimulated sustained apical Cl- secretion in monolayers of T84 cells without increasing intracellular cyclic nucleotides or Ca2+ levels. Tyrphostin 47 also stimulated Cl- secretion in T84 monolayers, although it was short-lived. Transfection experiments in 3T3 fibroblasts and IEC-6 intestinal cells utilizing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) showed that genistein and tyrphostin 47 stimulated 125I efflux only in CFTR-transfected cells and not in CFTR-negative cells. Thus genistein- and tyrphostin 47-stimulated Cl- secretion involved CFTR. Genistein also acted synergistically with the Ca2+- and protein kinase C-dependent acetylcholine analogue, carbachol, to stimulate Cl- secretion in T84 monolayers. However, the Cl- secretory response to saturating concentrations of the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin, or the guanosine 3',5'- cyclic monophosphate (cGMP) agonist, Escherichia coli heat-stable enterotoxin, was not further enhanced by genistein. Although the mechanism of activation of Cl- secretion is unclear, these data suggest that tyrosine kinase activity limits basal Cl- secretion in T84 cells and that inhibition of T84 cell tyrosine kinase(s) stimulates apical membrane Cl- secretion, most likely through activation of the CFTR-Cl- channel. Moreover, genistein does not itself act through cAMP or cGMP elevation but appears to share a common Cl- secretory pathway with cyclic nucleotide-dependent agonists, whereas it augments the secretory responses to a Ca2+- and protein kinase C-dependent agonist.

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KW - Escherichia coli heat-stable enterotoxin

KW - guanylate cyclase

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KW - tyrosine kinases

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