ACTIVATION OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATORBY 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 inv estigated the regulatory pathway responsible for cGMP-mediated Cl- sec retion in the human colonic carcinoma cell line Caco-2 using whole-cel l voltage clamp techniques. Cyclic GMP or cAMP induced a 5-fold increa se in Cl- conductance (g(Cl)) 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) inhibit or, KT5823, but was inhibited by the specific peptide inhibitor of the cAMP-dependent protein kinase A (PKA), PKI5-24. The stimulatory effec ts of cGMP and cAMP on g(Cl) were not additive. The cystic fibrosis tr ansmembrane conductance regulator (CFTR) is a Cl- channel that is regu lated by intracellular ATP and by cAMP-dependent phosphorylation. In o rder to determine whether CFTR was involved in the cGMP-dependent incr ease in g(Cl), we tested the effect of intracellularly injected anti-C FTR505-511 antibodies previously shown to inhibit CFTR function. Antib odies introduced into individual cells via the patch pipette completel y inhibited cGMP-dependent current activation. Cyclic GMP also failed to activate g(Cl) in cystic fibrosis cells. Taken together, these stud ies demonstrate that activation of the CFTR via PKA-dependent phosphor ylation accounts for the cGMP-mediated increase in Cl- secretion in Ca co-2 cells.