monensin and Cystic-Fibrosis

The role of CFTR in lysosome acidification was examined in CFPAC-1 pancreatic adenocarcinoma cells with the delta F508 mutation that were transduced with a retroviral vector (PLJ-CFPAC) or with the normal CFTR gene (CFTR-CFPAC). Steady-state lysosomal pHi in intact cells was lower in PLJ-CFPAC cells than CFTR-CFPAC cells (3.55 vs 3.80) and was not affected by cAMP or forskolin. Initial rates of ATP-dependent acidification of isolated lysosomes and steady-state ATP-dependent pHi were similar in both cell lines over a range of chloride concentrations and were not altered when cells were exposed to cAMP or to forskolin prior to preparation of lysosomes. These observations suggest that CFTR plays no role in acidification of lysosomes, possibly due to limited permeability of lysosomal membranes to chloride.

Topics: Adenocarcinoma; Adenosine Triphosphate; Bucladesine; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Colforsin; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Dextrans; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Lysosomes; Membrane Proteins; Monensin; Mutation; Pancreatic Neoplasms; Spectrometry, Fluorescence

Lysosomal enzyme secretion by skin fibroblasts derived from cystic fibrosis homozygotes and normal donors of the same range of age, sex and culture passage number were compared. No differences were detected between these groups in their rates of spontaneous lysosomal enzyme release. Incubation of either normal or cystic fibrosis fibroblasts with cystic fibrosis serum did not alter these secretion rates. The lysosomal enzyme secretion of both normal and cystic fibrosis fibroblasts responded similarly to incubation with NH4Cl, monensin, A23187, trifluoperazine or Cytochalasin B. It was concluded that lysosomal enzyme hypersecretion, which has previously been suggested to occur in cystic fibrosis, is unlikely to provide a reliable diagnostic test for the disease.

Topics: Acetylglucosaminidase; Ammonium Chloride; Cell Line; Cystic Fibrosis; Cytochalasin B; Fibroblasts; Hexosaminidases; Humans; Kinetics; Lysosomes; Monensin