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Am J Physiol Cell Physiol 262: C251-C256, 1992;
0363-6143/92 $5.00
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AJP - Cell Physiology, Vol 262, Issue 1 C251-C256, Copyright © 1992 by American Physiological Society

ARTICLES

cAMP-activated chloride conductance in the colonic cell line, Caco-2

C. E. Bear and E. F. Reyes
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.

In this study we investigated the properties of adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- efflux in Caco-2 monolayers by measuring 125I efflux rates from preloaded cells and using patch-clamp electrophysiology. The addition of a cocktail containing 100 microM dibutyryl cAMP (DBcAMP), 10 microM forskolin, and 1 mM 3-isobutyl-1-methylxanthine caused a significant (P less than 0.05) increase in the rate of 125I efflux. Dissipation of cell potential by adding valinomycin (4.5 microM) with 135 mM extracellular KCl reduced the cAMP-evoked 125I 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, PCl greater than PI greater than Pgluconate, 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.(ABSTRACT TRUNCATED AT 250 WORDS)


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