Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers.
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.
Sears
C L
CL
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA.
Firoozmand
F
F
Mellander
A
A
Chambers
F G
FG
Eromar
I G
IG
Bot
A G
AG
Scholte
B
B
De Jonge
H R
HR
Donowitz
M
M
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D019833:Genistein; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007529:Isoflavones / Q000494:pharmacology*; D009570:Nitriles / Q000494:pharmacology*; D009712:Nucleotides, Cyclic / Q000378:metabolism; D010636:Phenols / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D015290:Second Messenger Systems; D020032:Tyrphostins
C505032:CFTR protein, human; D018118:Chloride Channels; D002712:Chlorides; D007529:Isoflavones; D009570:Nitriles; D009712:Nucleotides, Cyclic; D010636:Phenols; D020032:Tyrphostins; C060152:tyrphostin 47; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D002118:Calcium