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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 -G...

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Published in:The Journal of biological chemistry 1996-06, Vol.271 (26), p.15542-15548
Main Authors: Seksek, O, Biwersi, J, Verkman, A S
Format: Article
Language:English
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Summary: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 ΔF508-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 HCO − 3 -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, ΔF508) (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, ΔF508) (not significant). Similarly, significant pH differences were not found for control versus CFTR-expressing cells in 25 m M HCO − 3 buffer. In epithelial cells, which do not express CFTR, trans -Golgi pH was (in 25 m M HCO − 3 ) 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 m M HCO − 3 ) 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 HCO − 3 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
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.271.26.15542