title: Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers.
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.
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Sears
authors.forename: F
authors.initials: F
authors.lastname: Firoozmand
authors.forename: A
authors.initials: A
authors.lastname: Mellander
authors.forename: F G
authors.initials: FG
authors.lastname: Chambers
authors.forename: I G
authors.initials: IG
authors.lastname: Eromar
authors.forename: A G
authors.initials: AG
authors.lastname: Bot
authors.forename: B
authors.initials: B
authors.lastname: Scholte
authors.forename: H R
authors.initials: HR
authors.lastname: De Jonge
authors.forename: M
authors.initials: M
authors.lastname: Donowitz
mesh_terms: 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
chemical_list: 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