Phenanthrolines – a new class of CFTR chloride channel openers

1. A number of phenanthrolines and benzoquinolines were examined for their ability to activate epithelial chloride secretion by measuring short circuit current (SCC) using the mouse colon epithelium. 1,10 phenanthroline stimulated electrogenic chloride secretion with an EC(50) of 612±10 μ and a Hill slope of 4.9±0.3. A similar pharmacology was demonstrated by both 1,7 and 4,7 phenanthrolines, 7,8 benzoquinoline and phenanthridine. 2. Evidence that the increase in SCC caused by 1,10 phenanthroline was due to chloride secretion is based upon (a) inhibition of the current by furosemide, (b) failure of cystic fibrosis (CF) colons to respond and (c) an associated net flux of (36)Cl(−). 3. 1,10 Phenanthroline affected neither the generation of cyclic AMP or the concentration of intracellular Ca(2+) in colonic epithelial cells. 4. 1,10 phenanthroline affected the chloride conductance of the apical membrane, as shown by an increase in chloride current in ‘apical membrane only' preparations in the presence of an apical to basolateral chloride gradient. The increase in chloride current was inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and was not present in CF colons. 5. Additionally, 1,10 phenanthroline activated basolateral K(+) channels, both Ca(2+)- and cyclic AMP-sensitive channels, as shown by inhibitor studies with charybdotoxin (ChTX) and XE991, and after the apical membrane was permeabilized with nystatin. 6. The phenanthrolines and benzoquinolines described here, with dual actions affecting CFTR and basolateral K(+) channels, may constitute useful lead compounds for adjunct therapy in CF

Digitoxin mimics gene therapy with CFTR and suppresses hypersecretion of IL-8 from cystic fibrosis lung epithelial cells

Cystic fibrosis (CF) is a fatal, autosomal, recessive genetic disease that is characterized by profound lung inflammation. The inflammatory process is believed to be caused by massive overproduction of the proinflammatory protein IL-8, and the high levels of IL-8 in the CF lung are therefore believed to be the central mechanism behind CF lung pathophysiology. We show here that digitoxin, at sub nM concentrations, can suppress hypersecretion of IL-8 from cultured CF lung epithelial cells. Certain other cardiac glycosides are also active but with much less potency. The specific mechanism of digitoxin action is to block phosphorylation of the inhibitor of NF-κB (IκBα). IκBα phosphorylation is a required step in the activation of the NF-κB signaling pathway and the subsequent expression of IL-8. Digitoxin also has effects on global gene expression in CF cells. Of the informative genes expressed by the CF epithelial cell line IB-3, 58 are significantly (P < 0.05) affected by gene therapy with wild-type (CFTR CF transmembrane conductance regulator). Of these 58 genes, 36 (62%) are similarly affected by digitoxin and related active analogues. We interpret this result to suggest that digitoxin can also partially mimic the genomic consequences of gene therapy with CF transmembrane conductance regulator. We therefore suggest that digitoxin, with its lengthy history of human use, deserves consideration as a candidate drug for suppressing IL-8-dependent lung inflammation in CF

Actions of adenosine A(1) and A(2) 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 A(1) and A(2) 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 A(1) receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A(2) receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the K(i) for A(1) 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