Malcolm M.C. Pereira

Sildenafil (Viagra) corrects DeltaF508-CFTR location in nasal epithelial cells from patients with cystic fibrosis.

Background: Most patients with cystic fibrosis (CF) have a ΔF508 mutation resulting in abnormal retention of mutant gene protein (ΔF508-CFTR) within the cell. This study was undertaken to investigate ΔF508-CFTR trafficking in native cells from patients with CF with the aim of discovering pharmacological agents that can move ΔF508-CFTR to its correct location in the apical cell membrane. Method: Nasal epithelial cells were obtained by brushing from individuals with CF. CFTR location was determined using immunofluorescence and confocal imaging in untreated cells and cells treated with sildenafil. The effect of sildenafil treatment on CFTR chloride transport function was measured in CF15 cells using an iodide efflux assay. Results: In most untreated CF cells ΔF508-CFTR was mislocalised within the cell at a site close to the nucleus. Exposure of cells to sildenafil (2 hours at 37°C) resulted in recruitment of ΔF508-CFTR to the apical membrane and the appearance of chloride transport activity. Sildenafil also increased ΔF508-CFTR trafficking in cells from individuals with CF with a single copy ΔF508 (ΔF508/4016ins) or with a newly described CF trafficking mutation (R1283M). Conclusions: The findings provide proof of principle for sildenafil as a ΔF508-CFTR trafficking drug and give encouragement for future testing of sildenafil and related PDE5 inhibitors in patients with CF.

An antibody against a cftr-derived synthetic peptide, incorporated into living submandibular cells, inhibits beta-adrenergic stimulation of mucin secretion

An antibody raised against a peptide in the first nucleotide-binding domain (NBD) of CFTR [1], incorporated into intact rat submandibular acini by hypotonic swelling, inhibited beta-adrenergic stimulated mucin secretion, without affecting cyclic AMP rise. The data are the first to show that a CFTR-antibody-containing cell results in defective stimulation of mucin secretion, as is seen in CF cells, and that this can be reversed by an excessive increase in cyclic AMP.

Benzo(c)quinolizinium drugs inhibit degradation of ΔF508-CFTR cytoplasmic domain

Proteins comprising the first nucleotide-binding- and R-domains of wild-type and Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) have been synthesised by in vitro transcription/translation. The kinetics and extent of degradation of wild-type and Delta F508 cytoplasmic domain proteins in rabbit reticulocyte lysates, in which proteasome activity was inhibited, were similar, with a half-life of approximately 4h. The results show for the first time, that the benzo(c)quinolizinium compounds, MPB-07 and MPB-91, selectively inhibit degradation of the Delta F508 cytoplasmic domain protein. Studies using protease inhibitors demonstrated that both Delta F508 and wild-type proteins are substrates for cysteine proteases. The studies provide evidence that benzo(c)quinolizinium compounds protect a proteolytic cleavage site by direct binding to the first cytoplasmic domain of Delta F508-CFTR and this is a likely mechanism for increasing Delta F508-CFTR trafficking in intact cells.

A cyclic nucleotide PDE5 inhibitor corrects defective mucin secretion in submandibular cells containing antibody directed against the cystic fibrosis transmembrane conductance regulator protein

A selective cyclic nucleotide PDE5 inhibitor corrected the defective mucin secretion response to the β‐agonist isoproterenol in submandibular acinar cells inhibited by antibody directed against the cystic fibrosis transmembrane conductance regulator. The PDE5 inhibitor was as effective as cpt‐cyclic AMP or a selective PDE4 inhibitor. However, the PDE5 inhibitor had no effect on basal or isoproterenol‐stimulated cyclic AMP levels and did not stimulate mucin secretion. The results showing, for the first time, correction of the CFTR mucin secretion defect by a PDE5 inhibitor, which may involve cyclic GMP, will have a major impact in development of a rational drug treatment for cystic fibrosis.

The CFTR-mediated protein secretion defect: pharmacological correction

Abstract. The cystic fibrosis transmembrane conductance regulator (CFTR) mediates secretion of mucins and serous proteins. The aim was to correct pharmacologically the CFTR defect in protein secretion in airway gland cells and so to correct the viscous mucous secretions in cystic fibrosis (CF) airways and lungs. The strategies tested included direct activation of CFTR, bypass of CFTR-mediated protein secretion and movement of the mutated form of CFTR (ΔF508-CFTR) to the cell membrane. Compounds related to 3-isobutyl-1-methylxanthine (IBMX), including a selective type-IV phosphodiesterase inhibitor and the adenosine receptor antagonists 8-cyclopentyltheophylline (CPT) and 8-cyclopentyl-1,3-dipropylxanthine (CPX), corrected the defective β-adrenergic stimulation of mucin secretion in CFTR antibody-inhibited submandibular gland cells. CPT also corrected lactoferrin secretion in ΔF508/ΔF508-CFTR nasal gland cells. The data suggest that correction of CFTR protein secretion activity is not mediated by excessive increase in cyclic AMP, involves direct interaction with CFTR but does not require increase in CFTR Cl– channel activity. Regulated glycoprotein secretion was characterised in the airway gland cell line Calu-3 to investigate whether a CFTR bypass is present. Studies of ΔF508-CFTR trafficking using confocal imaging showed that some ΔF508-CFTR colocalised with the apical membrane protein CD59; however a large amount was mislocalised within the cell. The results showing pharmacological correction of the defective CFTR-mediated protein secretion afford promise for the development of a rational drug therapy for CF patients.

Actions of adenosine A1 and A2 receptor antagonists on CFTR antibody‐inhibited β‐adrenergic mucin secretion response

1 The cystic fibrosis gene protein, the cystic fibrosis transmembrane conductance regulator (CFTR) acts as a chloride channel and is a key regulator of mucin secretion. The mechanism by which 3‐isobutyl‐1‐methylxanthine (IBMX) corrects the defect in CFTR mediated β‐adrenergic stimulation of mucin secretion has not been determined. The present study has investigated the actions of adenosine A1 and A2 receptor antagonists to determine whether ability to stimulate mucin secretion correlates with correction of CFTR antibody inhibited β‐adrenergic response and whether excessive cyclic AMP rise is required. 2 CFTR antibodies were introduced into living rat submandibular acini by hypotonic swelling. Following recovery, mucin secretion in response to isoproterenol was measured. 3 The adenosine A1 receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A2 receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the Ki for A1 receptor antagonism (10 nM) did not stimulate mucin secretion. 4 DMPX, although a potent stimulator of mucin secretion, did not correct CFTR antibody inhibited mucin secretion. 5 CPT corrected defective CFTR antibody inhibited mucin secretion at a high (1 mM) concentration, suggesting a mechanism other than adenosine receptor antagonism. 6 DMPX potentiated the isoproterenol induced cyclic AMP rise, whereas CPT did not. 7 Correction of the defective CFTR mucin secretion response did not correlate with ability to stimulate mucin secretion and did not require potentiation of β‐adrenergic induced increases in cyclic AMP. This affords real promise for the development of a selective drug treatment for cystic fibrosis.