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Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers
C L Sears1, F Firoozmand, A Mellander
1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA.
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 Ca(2+)- 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 Ca(2+)- and protein kinase C-dependent agonist.
Insights
Tyrosine kinase inhibitors like genistein stimulate epithelial cell chloride secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR) channel. This suggests tyrosine kinase activity normally limits basal chloride secretion.
Area of Science:
- Cell Biology
- Physiology
- Molecular Biology
Background:
- The role of tyrosine phosphorylation in regulating epithelial cell chloride (Cl-) secretion remains largely unknown.
- Understanding this mechanism is crucial for deciphering intestinal fluid balance and diseases like cystic fibrosis.
Purpose of the Study:
- To investigate whether tyrosine kinase activation is involved in the regulation of epithelial Cl- secretion.
- To determine the specific pathways and channels involved in tyrosine kinase-mediated Cl- secretion.
Main Methods:
- Utilized human intestinal T84 cells as a model for intestinal Cl- secretion.
- Employed tyrosine kinase inhibitors genistein and tyrphostin 47.
- Conducted transfection experiments in 3T3 fibroblasts and IEC-6 cells with wild-type cystic fibrosis transmembrane conductance regulator (CFTR) to assess channel involvement.
Main Results:
- Genistein and tyrphostin 47 stimulated apical Cl- secretion in T84 cells, independent of intracellular cyclic nucleotides or Ca2+.
- Stimulation of Cl- secretion by these inhibitors was dependent on CFTR expression, as shown by 125I efflux in transfected cells.
- Genistein synergized with carbachol but not with forskolin or E. coli heat-stable enterotoxin, indicating a distinct pathway from cAMP/cGMP agonists.
Conclusions:
- Tyrosine kinase activity appears to limit basal Cl- secretion in T84 cells.
- Inhibition of tyrosine kinases stimulates apical membrane Cl- secretion, likely via CFTR-Cl- channel activation.
- Genistein augments Ca2+-dependent secretion and shares a pathway with cyclic nucleotide-dependent agonists without directly elevating cAMP or cGMP.