Cystic fibrosis is a life-threatening, wide spread genetic disease diagnosed in 1 to 3000 livebirths of the Caucasian population. Here a mouse model for this disease is described and optimized using the CFTR-channel selective inhibitor CFTR(inh 172). The target parameter was mucociliary clearance measured using microdialysis of the transported fluorescent dye rhodamine in the mouse trachea in situ. The impact of Ap4A (diadenosine tetraphosphate) as a potential drug was investigated. Its inhalation was effective at low concentrations; established compounds such as Salbutamol and UTP increased mucociliary clearance as well. Our data show a functioning model of cystic fibrosis and the effectiveness of the newly tested Ap4A.
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is important for ion regulation. It represents a Cl− channel expressed in epithelial cells in e.g. airways, intestine, pancreas and testis. CFTR is also a regulator of other ion channels and other proteins [
CF treatment with mucoactive agents comprises three approaches: 1) expectorants, which add water to the airway; 2) ion transport modifiers, which promote ion and water transport across airway epithelium; and 3) mucokinetics, which improve cough-mediated clearance by increasing airflow or reducing sputum adhesivity [
Extracellular nucleotides regulate ion transport, ciliary beat frequency, mucociliary clearance and improve mucus (mucosal) hydration on the surface of the airways by acting on P2 nucleotide receptors, in particular the P2Y2 receptor [
Some novel agents and procedures to treat cystic fibrosis are summarized [
Not many CF animal models exist which show the typical symptoms of impaired mucociliary clearance. We decided to use the CFTR-channel selective inhibitor CFTR(inh 172), a thiazolidinone, being identified by high-throughput screening tested for blocking cholera toxin-induced intestinal fluid secretion [17,18]. By blocking the CFTR-chloride channel in the airways, CFTR(inh 172) can mimic the symptoms of the defective CFTR in cystic fibrosis [
The aim of this study was to use and optimize the CFTR inhibitor CFTR(inh 172) for a mouse model of CF and to test Ap4A in this model of CF as a P2X1-, P2X2-, P2X3-receptor agonist.
The CFTR(inh 172) was from Sigma-Aldrich, Steinheim, Germany. Ap4A, UTP, Salbutamol and D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) were purchased from Sigma-Aldrich.
CFRT(inh 172) may be used orally or i.p.; solubility problems must be overcome by e.g. dissolving it in DMSO (mice get 4 ml/kg b.w. DMSO in total) or in TPGS (D-α-Tocopherol polyethylene glycol 1000 succinate) [
Ap4A, UTP and Salbutamol were dissolved in 0.12% saline and nebulized using a Pari-Turbo-Boy (PARI GmbH, Starnberg, Germany) with a custom made mouse-adapter. Mice inhaled the mentioned compounds for 10 minutes before starting each experiment. For control experiments 0.12% saline was nebulized. It is known that approximately 7 mg of a 40 mg nebulizer load is deposited in the lungs of a human [
To study the mucociliary clearance in situ mice from a C57BL/6 strain (Charles River Laboratories, Sulzfeld, Germany) were used. Mice were allowed food and water ad libitum. All experiments were approved by the German animal welfare committee (8.87/5010.37.09.245).
Mucociliary clearance was determined as recently described [
For all experiments, CMA/20/04 PC probes and a CMA/102 microdialysis pump (CMA Microdialysis, Solna, Sweden) were used. The probe was perfused by phosphate buffered saline at a flow rate of constant 4 µL/min. After deposition of the fluorescent dye, the dialysate was collected in intervals of 15 s in 96-well Nunc plates with a conical bottom, to take account of the small sample volume of 1 µL per well. The experiment was finished after 24 min, when each of the 96 wells was filled with 1 µL dialysate. The plate was then rapidly inserted into a FluoStar Galaxy fluorescence microplate reader (BMG Lab Tech, 07743 Jena, Germany). The fluorescence intensity recorded as counts in each well was obtained as an equivalent of the dye concentration at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. In earlier test experiments recovery was independent of the dye concentration and it amounted 10% - 11% (data not shown). The death time of the probe with its tubes was obtained by placing the probe directly in a container with rhodamine dye and starting the perfusion immediately. In every performed experiment the dye was detectable in the 5th well (the 5th 15 s time interval), so the time to get the death volume out, here was 60 s. This 60 s were subtracted from each calculated dye travelingtime.
The data was collected using a standard personal computer with FluoStar Galaxy software (BMG Lab Tech, Jena, Germany). The first appearance of fluorescent dye in the MCT measurements was detected by averaging the mean background dye concentration and using the first measuring point, which was three SD above this mean background. More details were recently described [
The results are expressed as mean ± SEM of a given number of independent experiments. For statistical evaluation multiple comparisons of means were carried out by one-way analysis of variance followed by a post-hoc test (Student’s t-test).
Next mice were investigated which were pretreated with the CFTR(inh 172) to induce a model of CF. Both i.p. and oral administration of the inhibitor were used in order to find out optimum conditions.