Abstract
Cystic fibrosis (CF) is a syndrome caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. Despite advances in our understanding of the molecular pathogenesis of CF, the link between CFTR gene mutations and the pathogenesis of CF lung disease remains poorly defined. CFTR has been assigned a number of putative functions that may contribute to innate airway defense, including the regulation of adenosine 5'-triphosphate (ATP) release into the extracellular environment. Because extracellular ATP and uridine 5'-triphosphate (UTP) may regulate airway mucociliary clearance via interaction with luminal P2Y2 receptors, the loss of CFTR-mediated nucleotide release could explain the defect in CF airway defense. We tested the physiologic importance of CFTR-mediated nucleotide release in vivo by directly measuring levels of ATP and UTP in nasal airway surface liquid from normal and CF subjects. Because these basal nucleotide levels reflect the net activities of nucleotide release and metabolic pathways, we also measured constitutive rates of nucleotide release and metabolism on well-differentiated normal and CF airway cultures in vitro. The measurement of ATP release rates were paralleled by in vivo studies employing continuous nasal perfusion in normal and CF subjects. Finally, the regulation of ATP release by isoproterenol and methacholine-stimulated submucosal gland secretion was tested. These studies revealed that steady-state ATP and UTP levels were similar in normal (470 +/- 131 nM and 37 +/- 7 nM, respectively) and CF (911 +/- 199 nM and 33 +/- 12 nM, respectively) subjects. The rates of both ATP release and metabolism were also similar in normal and CF airway epithelia both in vitro and in vivo. Airway submucosal glands did not secrete nucleotides, but rather, secreted a soluble nucleotidase in response to cholinergic stimuli. The concentration of ATP in airway surface liquid is in a range that is relevant for the activation of airway nucleotide receptors. However, despite this finding that suggests endogenous nucleotides may be important for the regulation of mucociliary clearance, our data do not support a role for CFTR in regulating extracellular nucleotide concentrations on airway surfaces.
Highlights
Cystic fibrosis (CF) is a monogenetic disease characterized by chronic airway infection and bronchiectasis that is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene
No difference in uridine 5Ј-triphosphate (UTP) concentrations was detected in nasal airway surface liquid (ASL) from normal and CF subjects (37.0 Ϯ 7.2 nM and 33.3 Ϯ 11.8 nM, respectively; p ϭ 0.81; Fig. 1)
To determine whether the presence of extracellular nucleotides was the result of cellular damage, we measured lactate dehydrogenase (LDH) activity in samples
Summary
Cystic fibrosis (CF) is a monogenetic disease characterized by chronic airway infection and bronchiectasis that is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. A number of in vitro studies showed either direct ATP conduction through CFTR using patch clamp techniques, or evidence for a direct relationship between normal CFTR expression and ATP release [7,8,9,10,11,12,13,14]. Materials and Methods: We tested the physiologic importance of CFTR-mediated nucleotide release in vivo by directly measuring levels of ATP and UTP in nasal airway surface liquid from normal and CF subjects. Results: These studies revealed that steady-state ATP and UTP levels were similar in normal (470 Ϯ 131 nM and 37 Ϯ 7 nM, respectively) and CF (911 Ϯ 199 nM and 33 Ϯ 12 nM, respectively) subjects The rates of both ATP release and metabolism were similar in normal and CF airway epithelia both in vitro and in vivo. Despite this finding that suggests endogenous nucleotides may be important for the regulation of mucociliary clearance, our data do not support a role for CFTR in regulating extracellular nucleotide concentrations on airway surfaces
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