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 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)
Bear
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Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Reyes
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D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D018118:Chloride Channels; D002712:Chlorides / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology*; D004553:Electric Conductivity; D007455:Iodine / Q000493:pharmacokinetics; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000502:physiology; D013268:Stimulation, Chemical
D018118:Chloride Channels; D002712:Chlorides; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D005576:Colforsin; D003994:Bucladesine; D007455:Iodine; D000242:Cyclic AMP; D015056:1-Methyl-3-isobutylxanthine