EVIDENCE AGAINST DEFECTIVE TRANS-GOLGI ACIDIFICATION IN CYSTIC-FIBROSIS

Defective organelle acidification has been proposed as a unifying hypo thesis to explain the pleiotropic cellular abnormalities associated wi th cystic fibrosis. To test whether cystic fibrosis transmembrane cond uctance regulator (CFTR) participates in trans-Golgi pH regulation, in traluminal trans-Golgi pH was measured in stably transfected Swiss 3T3 fibroblasts (expressing CFTR or Delta F508-CFTR) and CFTR-expressing and nonexpressing epithelial cells. trans-Golgi pH was measured by rat io-imaging confocal microscopy using a liposome injection procedure to label the lumen of trans-Golgi with fluid phase fluorescein and rhoda mine 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 -2-oxa-1,3-diazol-4-yl)amino]caproyl)-sphingosine. In unstimulate d 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) a nd 6.23 +/- 0.06 (n = 60, Delta F508) (not significant). After stimula tion 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, Delta F508) (not s ignificant). Similarly, significant pH differences were not found for control versus CFTR expressing cells in 25 mM HCO3- buffer. In epithel ial cells, which do not express CFTR, trans-Golgi pH was (in 25 mM HCO 3-) 6.36 +/- 0.04 (n = 33) and 6.34 +/- 0.08 (n = 23, CPT-cAMP) in MDC K 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, tra ns-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 im aging in individual endosomes labeled with fluorescein-rhodamine dextr ans. Comparing control and CFTR expressing fibroblasts, average endoso me 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 indic ate that CFTR expression and activation do not influence pH in the tra ns-Golgi and endosomal compartments, providing direct evidence against the defective acidification hypothesis.