Activation of the cystic fibrosis transmembrane regulator by cyclic AMP is not correlated with inhibition of endocytosis.
Based on the observation (Bradbury et al. (1992) Am. J. Physiol. 262, C752-C759) that conditions known to activate the cystic fibrosis transmembrane regulator protein (CFTR) increase the rate of exocytosis and decrease the rate of endocytosis, it was proposed that activation of the CFTR may involved cAMP-dependent fusion of CFTR containing endosomes with the apical membrane. We have tested this hypothesis in two cell lines derived from epithelia that express defective chloride transport in cystic fibrosis (CF): the human colonic cell line, T84, and the tracheal cell line 9HTEo-. The dose-dependence of forskolin- and CPT-cAMP-induced inhibition of endocytosis were compared with the dose-dependence of chloride channel activation. Endocytosis was determined from the uptake of FITC-dextran, and assayed in purified endosomes. Chloride channel activity was measured from the rate of I-efflux. If the fusion hypothesis is correct: (1) concentrations of agonist that inhibit endocytosis should activate chloride channel activity, and (2) the relationship between endocytosis and channel activation should be similar for forskolin and CPT-cAMP. Results in both cell lines were inconsistent with these postulates, suggesting that either chloride channel activation and the inhibition of endocytosis are separate effects of cAMP, or that the increase in apical CFTR resulting from agonist-dependent inhibition of endosomal fusion is minimal.
Santos
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GF
Research Institute, Children's Hospital Oakland, CA 94609.
Reenstra
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WW
D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003911:Dextrans; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D006801:Humans; D007455:Iodine / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D013997:Time Factors
C505032:CFTR protein, human; D018118:Chloride Channels; D003911:Dextrans; D008565:Membrane Proteins; C015219:fluorescein isothiocyanate dextran; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D007455:Iodine; D000242:Cyclic AMP; D016650:Fluorescein-5-isothiocyanate