CAMP-ACTIVATED CHLORIDE CONDUCTANCE IN THE COLONIC CELL-LINE, CACO-2

In this study we investigated the properties of adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- efflux in Caco-2 monolayers by measuring I-125 efflux rates from preloaded cells and using patch-clamp electrophysiology. The addition of a cocktail containing 100-mu-M dibutyryl cAMP (DBcAMP), 10-mu-M forskolin, and 1 mM 3-isobutyl-1-methylxanthine caused a significant (P < 0.05) increase in the rate of I-125 efflux. Dissipation of cell potential by adding valinomycin (4.5-mu-M) with 135 mM extracellular KCl reduced the cAMP-evoked I-125 efflux. These results suggest that cAMP-stimulated anion efflux occurs through a conductive pore or channel. Whole cell currents evoked with DBcAMP or forskolin were anion selective, P(Cl) > P(I) > P(gluconate), and exhibited a linear current-voltage (I-V) relationship. Currents evoked with depolarizing or hyperpolarizing voltage steps showed no evidence of time-dependent activation or inactivation. Single Cl- channels were stimulated in cell-attached patches after treatment with cAMP. Onset of channel activity occurred after 20-30s of cAMP treatment, and the response was long lasting. The I-V relationship for the channel activated in cell-attached patches by cAMP was best fit using two linear regressions. The slope conductance of the channel was 3.2 +/- 0.6 and 7.4 +/- 0.3 pS at hyperpolarizing and depolarizing potentials, respectively. Substitution of 140 mM NaCl with 70 mM NaCl in the patch pipette resulted in a positive shift in reversal potential, indicating that the channel is anion selective. The channel exhibited a linear I-V relationship upon patch excision when similar Cl- concentrations (140 mM) existed in both pipette and bath solutions with a slope conductance of 7.9 +/- 0.5 pS. These results suggest that the increase in Cl- secretion stimulated by intracellular cAMP accumulation in Caco-2 cells may be due to the activation of this 7.9-pS Cl- channel.