Evidence against defective trans-Golgi acidification in cystic fibrosis

Abstract

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 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, 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 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.