Evidence against defective trans-Golgi acidification in cystic fibrosis.
Defective organelle acidification has been proposed as a unifying hypothesis to explain the pleiotropic cellular abnormalities associated with cystic fibrosis. To test whether cystic fibrosis transmembrane conductance regulator (CFTR) participates in trans-Golgi pH regulation, intraluminal trans-Golgi pH was measured in stably transfected Swiss 3T3 fibroblasts (expressing CFTR or DeltaF508-CFTR) and CFTR-expressing and nonexpressing epithelial cells. trans-Golgi pH was measured by ratio-imaging confocal microscopy using a liposome injection procedure to label the lumen of trans-Golgi with fluid phase fluorescein and rhodamine chromophores (Seksek, O., Biwersi, J., and Verkman, A. S.(1995) J. Biol. Chem. 270, 4967-4970). Selective labeling of trans-Golgi was confirmed by colocalization of the delivered fluid phase fluorophores with N-(6-[(7-nitrobenzo-2-oxa-1, 3-diazol-4-yl)amino]caproyl)-sphingosine. In unstimulated fibroblasts in HCO3--free buffer, trans- Golgi pH was 6.25 +/- 0.04 (mean +/- S.E.; n = 80, vector control), 6.30 +/- 0.03 (n = 74, CFTR) and 6.23 +/- 0.06 (n = 60, DeltaF508) (not significant). After stimulation of plasma membrane Cl- conductance by 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), trans-Golgi pH was 6.42 +/- 0.07 (n = 22, control), 6.47 +/- 0.07 (n = 20, CFTR), and 6.35 +/- 0. 07 (n = 22, DeltaF508) (not significant). Similarly, significant pH differences were not found for control versus CFTR-expressing cells in 25 mM HCO3- buffer. In epithelial cells, which do not express CFTR, trans-Golgi pH was (in 25 mM HCO3-) 6.36 +/- 0.04 (n = 33) and 6.34 +/- 0.08 (n = 23, CPT-cAMP) in MDCK cells and 6.25 +/- 0.04 (n = 18) and 6.24 +/- 0.06 (n = 15, CPT-cAMP) in SK-MES-1 cells. In Calu-3 cells, which natively express CFTR, trans-Golgi pH was (in 25 mM HCO3-) 6.19 +/- 0.05 (n = 25) and 6.17 +/- 0.08 (n = 23, CPT-cAMP). To test whether CFTR expression affects pH in the endosomal compartment in HCO3- buffer, pH was measured by ratio imaging in individual endosomes labeled with fluorescein-rhodamine dextrans. Comparing control and CFTR-expressing fibroblasts, average endosome pH (range, 5.40-5.53 after 10 min; 4.79-4.89, 30 min) differed by <0.13 unit, both before and after cAMP stimulation. These results indicate that CFTR expression and activation do not influence pH in the trans-Golgi and endosomal compartments, providing direct evidence against the defective acidification hypothesis.
Seksek
O
O
Department of Medicine, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA.
Biwersi
J
J
Verkman
A S
AS
D016475:3T3 Cells; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D002417:Cattle; D002451:Cell Compartmentation; D002478:Cells, Cultured; D018118:Chloride Channels / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009154:Mutation; D011994:Recombinant Proteins; D014162:Transfection
D001639:Bicarbonates; C505032:CFTR protein, human; D018118:Chloride Channels; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin