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Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells

¹ Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
² Laboratoire physiologie des régulations cellulaires, UMR6558, Université de Poitiers, 40 avenue du recteur Pineau, 86022 Poitiers, France
³ Laboratoire de chimie organique, Faculté de médecine et de pharmacie de Poitiers, 34 rue du jardin des plantes, 86005 Poitiers, France
⁴ Department of Physiological Sciences, University Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK
⁵ Laboratorio di genetica molecolare, Istituto Giannina Gaslini, 16148 Genova, Italy
⁶ Department of Child Health, University Hospital of Wales, Heath Park, Cardiff, CF14 4XN, UK

View larger version (13K): [in a new window]   Fig. 1. Activation of CFTR-mediated iodide efflux by cAMP agonists in IB3-1 cells. Iodide efflux experiments were carried out following a 2 hour incubation at 37°C with 250 µM MPB compound (closed symbols) or not (open symbols). (A,B) A cAMP elevating cocktail (Cock: 10 µM forskolin, 500 µM cpt-cAMP, 500 µM IBMX) stimulated iodide efflux after MPB-07 (A) or MPB-91 (B) treatment (n=8 for each). (C) Bar graphs of the cAMP-dependent iodide efflux in IB3-1 cells after MPB treatment (filled bars).*P<0.05; ***P<0.001.

View larger version (19K): [in a new window]   Fig. 2. Activation of CFTR-mediated iodide efflux by MPB-91 in IB3-1 cells. Iodide efflux experiments were carried out following a 2 hour incubation in the loading solution at 37°C with 250 µM MPB-91 compound (closed symbols) or not (open symbols). (A) MPB-91 (250 µM) stimulated iodide efflux only in MPB-treated cells (n=8 for each). (B) Bar graphs presenting the iodide efflux in IB3-1 cells after MPB treatment (filled bars). ***P<0.001.

View larger version (14K): [in a new window]   Fig. 3. Effect of MPB-91 on the calcium and swelling activated iodide efflux in IB3-1 cells. (A) Averaged iodide efflux curves showing the lack of effect of MPB-91 treatment on calcium-induced-iodide efflux (1 µM A23187 was used, n=4). (B) Demonstration of a small decrease of the volume-sensitive chloride transport observed after MPB-91 treatment (a 50% hypo-osmotic solution was present from the time indicated by the arrow to the end of the experiment, n=4). (C) Bar graphs presenting the effects of MPB-91 treatment on calcium and volume-sensitive chloride transport in IB3-1 cells. Arrows indicate the time of drug additions. NS, non-significant difference; *P<0.05.

View larger version (41K): [in a new window]   Fig. 4. Immunoprecipitation and in vitro phosphorylation of CFTR in IB3-1 cells. (A) Location of band-B and -C forms are shown together with the positions of molecular weight markers. Immunoprecipitation and in vitro phosphorylation of non-mutated CFTR from Calu-3 cells (lane 2) and delF508 from IB3-1 cells treated for 2 hours with 250 µM MPB-91 (+MPB-91, lane 5) or without treatment (–MPB-91, lane 4). Lanes 1 and 3 are control experiments without CFTR antibody. (B) Densitometric measurement of CFTR after in vitro phosphorylation. Percentage of fully processed band C and B are shown after a 2 hour treatment of IB3-1 cells with 250 µM MPB-91 (+MPB-91) or without treatment (–MPB-91).

View larger version (69K): [in a new window]   Fig. 5. Characteristics of cells from nasal brushings. (A) Single cell stained with Giemsa/May-Grünwald, showing cilia at apical end. (B) Immunofluorescent dual labelling of cytokeratin (green) and CFTR (red) in cells from a non-cystic fibrosis individual. (C,D) Immunofluorescent dual labelling of CFTR (C) and CD59 (D) showing the same cells, viewed with appropriate filter, from a non-cystic fibrosis individual. Results are representative data from five non-cystic fibrosis individuals. Magnification x1000.

View larger version (17K): [in a new window]   Fig. 6. Effect of MPB-07 on confocal immunofluorescent labelling of CFTR in non-cystic fibrosis and delF508/delF508 cystic fibrosis epithelial cells from nasal brushings. Images show CFTR immunofluorescence in green with the nucleus counterstained with propidium iodide (red). (A,B) Non-cystic fibrosis cells. (C-J) Cystic fibrosis cells. (A,C,E,G,I) Untreated cells. (B,D,F,H,J) Cells treated for 2 hours at 37°C with MPB-07 (250 µM). Results are representative and show the predominant cell type from five non-cystic fibrosis individuals and from four delF508/delF508 cystic fibrosis individuals with or without MPB-07 treatment with the exception of I. This shows an example of delF508-CFTR present throughout the cell, a pattern of distribution present in less than 10% of untreated cells, although in this cystic fibrosis individual, the predominant cell type had a perinuclear delF508-CFTR location similar to that seen in C,E,G. Magnification x1000.

View larger version (9K): [in a new window]   Fig. 7. Quantification of the effect of MPB-07 on location of wild-type and delF508-CFTR in epithelial cells from nasal brushings. Cells were examined (up to 140 cells from each brushing) and categorised as having a distinct CFTR location of either predominantly apical (filled bars) or perinuclear (open bars). A and B show wild-type cells, C and D show delF508/delF508 cells; A and C are untreated, and B and D are MPB-07 treated. Cells showing intermediate distributions that were either a discrete location adjacent to the nucleus, characteristic of delF508 cells (juxtanuclear), or more evenly distributed throughout the cell including the apical region were counted but were not included in the histogram for clarity (see text). The data are mean±s.e.m. for cells from five non-cystic fibrosis and four delF508/delF508 cystic fibrosis individuals (>100 cells counted). *P<0.001; **P<0.0001 for difference from untreated delF508 cells as assessed by Student’s t-test.

View larger version (78K): [in a new window]   Fig. 8. Confocal immunofluorescent labelling of delF508-CFTR in cultured surface nasal epithelial cells from a delF508/delF508 cystic fibrosis individual. (A,B) XY confocal sections <1 µm from the apical surface. (C,D) XY confocal sections 7 µm from the apical surface. (E,F) XZ scans. A, C and E are untreated cells. B, D and F are cells treated for 18 hours at 37°C with MPB-07 (500 µM). Results are representative of data from three delF508/delF508 cystic fibrosis individuals. Magnification x2500.

View larger version (20K): [in a new window]   Fig. 9. Effect of MPB-91 on confocal immunofluorescent labelling of CFTR in F508/G551D cystic fibrosis epithelial cells from nasal brushings. Images show CFTR immunofluorescence in green with the nucleus counterstained with propidium iodide (red). A-D are untreated cells; E and F are cells treated for 2 hours at 37°C with MPB-91 (250 µM). The bar graph shows quantification of the effect of MPB-91 on CFTR location. 80 cells were examined from each condition and categorized as having a distinct CFTR location of either perinuclear (PN), juxtanuclear (JN), more evenly distributed (ED) throughout the cell including the apical region, or predominantly apical (Apical) as described in the text. Open bars are untreated cells; filled bars are cells treated with MPB-91.