ABSTRACT
Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl(-) transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 muM lubiprostone was -5.8 +/- 2.1 mV (CF, n = 12), -8.1 +/- 2.6 mV (C57Bl/6 wild-type, n = 12), and -5.3 +/- 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 muM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia.
Subject(s)
Alprostadil/analogs & derivatives , Chlorides/metabolism , Cystic Fibrosis/metabolism , Epithelium/drug effects , Epithelium/metabolism , Respiratory System/metabolism , Adenosine Triphosphate/pharmacology , Administration, Topical , Alprostadil/administration & dosage , Alprostadil/pharmacology , Animals , Biological Transport/drug effects , CLC-2 Chloride Channels , Chloride Channels/genetics , Chloride Channels/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Dose-Response Relationship, Drug , Electric Conductivity , Gene Expression Regulation/drug effects , Lubiprostone , Membrane Potentials/drug effects , Mice , Mice, Knockout , RNA, Messenger/genetics , RNA, Messenger/metabolism , Subcellular Fractions/drug effects , Subcellular Fractions/metabolismABSTRACT
Vascular permeability plays a key role in a wide array of life-threatening and sight-threatening diseases. Vascular endothelial growth factor can increase vascular permeability. Using a model system for nonproliferative diabetic retinopathy, we found that pigment epithelium-derived factor (PEDF) effectively abated vascular endothelial growth factor-induced vascular permeability. A 44-amino acid region of PEDF was sufficient to confer the antivasopermeability activity. Additionally, we identified four amino acids (glutamate-101, isoleucine-103, leucine-112, and serine-115) critical for this activity. PEDF, or a derivative, could potentially abate or restore vision loss from diabetic macular edema. Furthermore, PEDF may represent a superior therapeutic approach to sepsis-associated hypotension, nephrotic syndrome, and other sight-threatening and life-threatening diseases resulting from excessive vascular permeability.
