1. Introduction
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 [1]. Mutations of CFTR gene may cause cystic fibrosis (CF), the most common lethal autosomal recessive genetic disease in Caucasian population with a frequency of approx. 1 in 3000 livebirths [1] and 40,000 diseases in the EU. As observed in human CF patients and CF mouse models CFTR has an impact on lung, intestinal and on pancreatic fluid transport and on male fertility. The disturbed Cl− transport leads to increased salt concentrations in the respiratory system which reduces the function of antibacterial molecules from epithelial cells such as defensins. CF lung disease (mutation) is characterized by persistent pulmonary infection and mucus plugging of the airways initiated by the failure of solute transport across the airway epithelium [2].
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 [3]. The major aim, however, should be strategies correcting the ion transport deficiency of CF. Airway surfaces hydration can be obtained by stimulating secretion (through activation of the CFTR and/or calcium-activated chloride channels), and/or inhibiting fluid absorption (through the epithelial sodium channel) which finally results in stimulating mucociliary clearance which would lower secondary lung infection.
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 [4]. As demonstrated by in situ hybridization, the P2Y2 receptor mRNA is located in lung epithelial cells and not in smooth muscle or stromal tissue [5]. P2Y2 receptor agonists bypass the defective CFTR chloride channel by activating an alternative chloride channel [6]. They also inhibit sodium absorption, restore chloride conductance and rehydrate the CF airway surface liquid. Typical effective P2Y2 receptor agonists are UTP, ATP, UTPγS and ATPγS. UTP has an effect on ciliary beat frequency [7,8], also acts via P2Y4 receptors (PPADS is an antagonist) and has a strong effect on Ca2+ and Cl− currents [9]. Close to the pharmacological profile of UTP is Denufosol tetrasodium (INS37217) [6,10,11] which is going to be clinically developed but may be not much more effective than placebo with respect to lung function. A single inhalative administration of metabolically stable denufosol enhanced mucus transport for at least 8 h [10] being an advantage over other P2Y2 agonists.
Some novel agents and procedures to treat cystic fibrosis are summarized [12]: anti-infective drugs to be nebulized, hyperosmolaric agents, CFTR-assist drugs (they assist or repair the CFTR protein), ENaC (epithelial sodium channel) blockers and EPAC (cAMP-derivative stimulating like 8-pCPT-2’-O-Me-cAMP). A drug named Moli1901 was shown to increase Cl− secretion via an alternative chloride channel [13]. P2Y6 agonists [14] increase Ca2+ and thereby CFTR-dependent Cl− secretion. A3 receptor agonists were shown to enhance mucociliary clearance probably through Ca2+-mediated stimulation of ciliary motility of airway epithelium [15]. Anion and liquid secretion are essential for normal mucociliary transport defined by using bumetanide and dimethylamiloride [16]. Also a new causal therapy was recently established: ivacaftor, a selective potentiator of CFTR protein if a G551D mutation of the CFTR gene is the reason.
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 [19].
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.
2. Materials and Methods
2.1. Compounds
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.
Solubility of Compounds
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) [18] for either i.p. or oral application with an orogastric feeding needle. In all experiments the drug was given three times (24 h, 12 h and 3 h) before starting the experiment. For control experiments we used TPGS and DMSO alone, respectively.
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 [6].
2.2. Animals
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).
2.3. Mucociliary Clearance and in Situ Microdialysis
Mucociliary clearance was determined as recently described [20]. To measure the mucociliary transport, the mice were anesthetized with Avertin® (0.4 g/kg tribromethanol and 0.4 mL/kg amylalcohol) and body temperature was maintained at 37˚C by a heating pad in combination with a thermo-controller and a temperature probe (CMA Microdialysis, Solna, Sweden). For measurements of the tracheal mucociliary clearance, the trachea was unveiled carefully and a small incision was made directly beyond the larynx. A microcapillary tube (DETAKTA, 22,851 Norderstedt, Germany) with a diameter of 80 µm was loaded with Rhodamine 123 fluorescent dye (15 nL/g body weight) and introduced 16 mm into the trachea. The tube was connected to a micro syringe, so that the dye could easily be placed by pressing 500 nL air into the tube. To prevent a capillary suction effect of the surface of the tube, resulting in immediate detection of the dye, a small silicon sphere (<100 µm) was placed at the edge of the tube. Leaving the tube in its place, the tip of the microdialysis probe was inserted 4 mm through the same incision into the trachea and fixed in its position by a custom made retaining jig. It was important, not to let air come through the incision by the breathing animal, getting the airways dry and resulting in no measurable transport of the dye. Hence the incision has to be as small as possible, fitting probe and tube. After placing the dye, the mucociliary transport velocity could be calculated from the time, the dye needed to travel the defined distance of 12 mm through the trachea, to reach the tip of the microdialysis probe.
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 [21].
2.4. Statistical Analysis
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).
3. Results
3.1. Elaboration of Optimum Conditions for Use of CFTR(inh 172)
Figure 1 shows the effect of inhaled UTP (10 mM) and Ap4A (5 mM) on mucociliary transport (MCT) in healthy mice. The data indicate that both compounds are effective in accelerating the mucociliary transport velocity (compare data shown later for CFTR-inhibitor pretreated mice).
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.
Figure 2 shows a basic experiment indicating that i.p. administration of the CFTR(inh 172) acutely restrains mucociliary transport. A higher dose of 3 times 1 mg i.p.