Activation of the cystic fibrosis transmembrane conductance regulator by cGMP in the human colonic cancer cell line, Caco-2.
Intestinal chloride (Cl-) secretion can be induced by the heat-stable enterotoxin (STa) from Escherichia coli via generation of cGMP. We investigated the regulatory pathway responsible for cGMP-mediated Cl- secretion in the human colonic carcinoma cell line Caco-2 using whole-cell voltage clamp techniques. Cyclic GMP or cAMP induced a 5-fold increase in Cl- conductance (gCl) in the presence of intracellular ATP and 3-isobutyl-1-methylxanthine. Current activation by cGMP persisted in the presence of the type I cGMP-dependent protein kinase (PKG) inhibitor, KT5823, but was inhibited by the specific peptide inhibitor of the cAMP-dependent protein kinase A (PKA), PKI5-24. The stimulatory effects of cGMP and cAMP on gCl were not additive. The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by intracellular ATP and by cAMP-dependent phosphorylation. In order to determine whether CFTR was involved in the cGMP-dependent increase in gCl, we tested the effect of intracellularly injected anti-CFTR505-511 antibodies previously shown to inhibit CFTR function. Antibodies introduced into individual cells via the patch pipette completely inhibited cGMP-dependent current activation. Cyclic GMP also failed to activate gCl in cystic fibrosis cells. Taken together, these studies demonstrate that activation of the CFTR via PKA-dependent phosphorylation accounts for the cGMP-mediated increase in Cl- secretion in Caco-2 cells.
Tien
X Y
XY
Department of Medicine, University of Chicago, Illinois 60637.
Brasitus
T A
TA
Kaetzel
M A
MA
Dedman
J R
JR
Nelson
D J
DJ
D018118:Chloride Channels / Q000378:metabolism; D003110:Colonic Neoplasms; D006152:Cyclic GMP / Q000494:pharmacology*; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D014407:Tumor Cells, Cultured
C505032:CFTR protein, human; D018118:Chloride Channels; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011494:Protein Kinases; D006152:Cyclic GMP