Effect of P2X7 Receptor Knockout on AQP-5 Expression of Type I Alveolar Epithelial Cells

P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis.


Introduction
Alveolar epithelial type I (AT I) cells contribute 7% of total lung cells and cover over 95% of the alveolar surface. This thin epithelium allows the easy diffusion of gases and forms a barrier against the indiscriminate leakage of fluid into alveolar spaces. It also regulates the exchange of physiologically important solutes and water between circulating blood and the alveolar space. There is new evidence that AT I cells, in addition to alveolar epithelial type II (AT II) cells, contribute to active Na + transport across the alveolar epithelium. They contain functional ion channels such as amiloride-sensitive epithelial Na + channels (ENaC), cystic fibrosis transmembrane regulator (CFTR) and the water channel aquaporin-5 (AQP-5) [1]. Adenosine -59-phosphate (ATP)-gated purinergic receptors (P2XRs), P2X7R and P2X4R were also identified in AT I cells [2]. These channels are extracellular ligand-gated ion channels. The ion channel is for mono-and divalent cations (Na + , K + , Ca 2+ ) permeable and contains three functional domains: an extracellular domain that binds a native agonist, a transmembrane domain forming an ion channel pore, and an intracellular domain critical for regulation and crosstalk with various signaling pathways. Purinergic receptor signaling is involved in regulating many physiological and pathophysiological processes [3,4].
The P2X7R, a member of the ionotropic P2X receptor family is selectively localized in AT I cells and plays probably an important part in mediating extracellular ATP signalling [5]. Recent animal studies have identified the importance of P2X7R and pannexin-1 complex in interleukin-(IL) 1b maturation, lung inflammation and development of pulmonary fibrosis [6].
Pulmonary fibrosis is characterized by inflammation and fibrosis of the interstitium and destruction of alveolar histoarchitecture leading finally to a fatal impairment of lung function. Fibroblasts and alveolar epithelial cells seem to be the principal players in this pathological process and interact in a paracrine fashion [7]. Profibrotic cytokines such as transforming growth factor beta-1 (TGF-b1), platelet-derived growth factor (PDGF) and tumor necrosis factor alpha (TNF-a) are released causing fibroblast transformation, proliferation and accumulation of an extracellular matrix. Alveolar epithelial cells undergo i: increased epithelial cell death [8] and ii: several altered phenotypes e.g. AT II hyperplasia and epithelial mesenchymal transition (EMT) [9]. Injured alveolar epithelial cells in turn regulate fibroblasts and the activation of other lung cells. Surprisingly, knockout animals with deletion of AT I cell-specific antigens exhibit several pulmonary defects [10,11]. Data about the pulmonary phenotype of P2X7R-deficient mice are missing. Recently, it was shown that bleomycin (BLM)treated P2X7R-deficient mice have less neutrophil airway influx and inflammatory cytokine production with reduced pulmonary fibrosis in terms of lung collagen and matrix-remodeling proteins [6]. Water channels (aquaporins (AQPs)) are a family of waterspecific membrane channel proteins found in cellular membranes. Four channels (AQP-1, AQP-3, AQP-4 and AQP-5) are expressed in the respiratory tract, with predominantly non-overlapping cellular and subcellular distributions [12] (with original references cited therein). The main AQPs in peripheral lung are AQP-5 in AT I cells and AQP-1 in endothelial cells. AQP-5, cloned originally from salivary gland [13], is an aquaporin with yet unknown molecular function in the lung. Under pathological circumstances increased or decreased levels of AQP-5 protein were found. It was shown that BLM-induced pulmonary inflammation in rats is associated with an increase of the expression of AQP-5 [14]. Gabazza et al. (2004) [15] demonstrated that lung fibrosis, the end-stage of a chronic process in the lung is associated with decreased protein and mRNA expression of AQP-5.
Our own in vitro experiments have demonstrated that BLMtreated alveolar epithelial cells show an increase in the expression of P2X7R, calmodulin (CaM) and PKC-b1. From this we predict that increased Ca 2+ concentration stimulates PKC-b1, whereas the prerequisite for activating PKC-b1 after P2X7R increase remained to be determined [22].
As far as we know, a direct functional relationship between AQP-5 and P2X7R has not been established. Since AQP-5 and P2X7R are known to be present in AT I cells, we examined whether P2X7R knockout influences its pulmonary phenotype, particularly the AQP-5 expression. Further, we were interested in the involvement of AQP-5 in early processes of BLM-induced lung injury in P2X7R knockouts using precision cut lung slices and the immortal lung cell lines E10 and MLE-12.

Ethics Statement
All animal experiments were approved by the ethics committee of the Dresden University of Technology and the license for removal of organs was provided by the Landesdirektion Dresden (file no. 24-9168. 24-1/2007-26; file no. 24-9168.24-1/2010-11).

Mice
Wild type (C57BL/6) and P2rx7 2/2 mice (B6.129P2-P2rx7 tm1Gab /J) were obtained from Pfizer (New York, NY, USA) [23]. Our animals were housed at Animal Care Facility at the Medical Faculty ''Carl Gustav Carus'' of Dresden University of Technology. The mice had always free costant access to standard chow and water. All performed procedures were in accordance with the Technical University Dresden Animal Care and Use Committee Guidelines. For our experiments we examined lung tissues from male and female mice at different ages: Younger mice were aged from 2 to 3 months while older mice were aged from 11 to 12 months. Mice were anesthetized with intraperitoneal lethal dose of 5% (w/v) thiopental (Thiopental 0.5 g, Inresa, Freiburg, Germany) or killed by cervical fracture, followed by lung removal.  Herzog et al. (1996) [24]) were kindly provided by M. Williams (Pulmonary Center, Boston University School of Medicine, Boston, MA, USA). For cultivation DMEM/ Ham's F12 medium was purchased from Gibco (Life Technologies, Carlsbad, CA, USA), which was supplemented with 5% (v/v) fetal bovine serum (PAN Biotech GmbH, Aidenbach, Germany) and 2.5 mM L-glutamine (Merck Millipore, Billerica, MA, USA). E10 cells were grown at 37uC in a 5% CO 2 atmosphere up to a confluence of approximately 60-80%. They were seeded at a density of 3610 4 cells/ml and passaged three times per week (up to 25 passages). The mouse lung epithelial cell line MLE-12 [25] was cultured in complete HITES medium [26] containing: DMEM/Ham's F12 (1:1) (Life Technologies) supplemented with 4.5 mM L-glutamine (Merck Millipore), 2% (v/v) fetal bovine serum (Merck Millipore), 10 mM HEPES (Life Technologies), 0.005 mg/ml insulin, 0.01 mg/ml transferin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM beta-estradiol (Sigma-Aldrich, St. Louis, MO, USA) and 1% (v/v) penicillin/streptomycin (Merck Millipore). Cells were cultured at 37uC in a 5% CO 2 atmosphere up to 60-80% confluence and were seeded at a density of 6610 4 cells/ml. Cells were passaged continously two times a week to a maximum of 30 passages. For BLM treatment medium was completed by 100 mU/ml BLM (Cell Pharm GmbH, Bad Vilbel, Germany). Further we used oxATP (100 mM) from Sigma-Aldrich.

Immunohistochemistry
Embedding in paraffin, sectioning and dehydration were performed as already described above in 2.5. According to Kasper and Fehrenbach (2000) [30] by following the manufacturer's instructions of Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) slides were microwave irradiated 265 min at 1000 W in 0.01 M sodium citrate buffer (ph 6.0) followed by cooling down for 15 min. Slides were 26 rinsed with destilled water and treated with 0.3% hydrogen peroxide blocking solution for 30 min at RT. Afterwards slides were washed 265 min with PBS and blocked in 1.5% (v/v) goat serum for 20 min at RT. Subsequently the primary antibody was diluted in PBS and incubated overnight at 4uC. Used antibodies are shown in Table 1. Next day slides were washed 265 min in PBS and incubated with secondary biotinylated antibody for 45 min at RT. After washing 265 min in PBS, slides were incubated for 45 min at Vectastatin ABC Reagent at RT, followed by 265 min of washing in PBS. Afterwards sections were stained with 3,39-Diaminobenzidine (DAB) for 5 min and washed with destilled water for 265 min. The following counterstaining was done with hematoxylin for

RNA Isolation and Real-time Reverse Transcription PCR (Real-time RT PCR)
Isolation of total RNA was performed by using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the supplier's instructions. RNA concentrations were determined with Nano-Photometer (Implen, Munich, Germany). For digesting DNA DNase I (ThermoFisher Scientific, Waltham, MA, USA) was applied. Synthesis of cDNA was done with Revert Aid H Minus First Strand Synthesis Kit (ThermoFisher Scientific) from 1-5 mg of total RNA using oligo-(dT) 18 primers. Using CFX96 Real-Time PCR Detection System (Bio-Rad, Herkules, USA) and Maxima SYBR Green/Fluorescein qPCR Master Mix (ThermoFisher Scientific) for running PCR, the following conditions for all primer sets were used: initial denaturation 8 min at 95uC, followed by 40 amplification cycles (95uC for 20 sec; 58uC for 45 sec; 72uC for 20 sec) with a final extension step at 72uC for 2 min. Primers applied for real-time RT PCR are listed in Table 2. Identity of PCR products was proven by melting point analysis and agarose gel electrophoresis. Relative quantification of gene expression was done with the DDCT method using CFX Manager (Bio-Rad) with Polr2a and Rpl32 as housekeeping genes.

Precision-cut Lung Slices and Tissue Culture
Following the preparation procedure of precision-cut lung slices (PCLS) described by Held et al. (1999) [31] tissue preparation was done with several modifications. Wild type and P2rx7 2/2 mice up to an age of 3 months were anesthetized with an overdose of 5% (w/v) thiopental (Thiopental 0.5 g, Inresa, Freiburg, Germany) including 600 international units (IU) heparin (Ratiopharm, Ulm, Germany) and exsanguinated by cutting the left renal artery. The chest wall was opened and the lungs were perfused with 10 ml of room tempered PBS through the right ventricle. Subsequently the trachea was cannulated with a 20-gauge catheter (Saf-T-Intima with Y adapter, BD Biosciences, Franklin Lakes, NJ, USA) and the lungs were filled with 1% (w/v) low-melting agarose (Sigma-Aldrich, St. Louis, MO, USA) solved in PBS at 37uC in situ. For agarose solification the mouse cadaver was cooled with ice for 15 min at 4uC. Next, solidified lungs were removed and separated into the different lobes. Each lobe was embedded in 3% (w/v) prewarmed agarose at 42uC. For gelling, agarose was cooled with ice at 4uC for 15 min. Afterwards each lobe was cut with Krumdieck tissue slicer (Alabama Research and Development, Munford, AL, USA) into 500 mm thick slices using DMEM/F12 medium with 15 mM Hepes and 2.5 mM L-glutamine (Life Technologies, Carlsbad, CA, USA) cooled down to 4uC. Then tissue slices were transferred into cell culture plates containing above-mentioned medium including 1% (v/v) pen/strep (Merck Millipore) and 0.5% (v/v) FCS (PAN Biotech GmbH) at 37uC. During the following 3 h medium was changed every 30 min. For add-on tissue culture two lung slices each were transferred on one Teflon mesh insert (Roller Insert Type A, Vitron Inc, Tucson, AZ, USA) and placed into a 20 ml glass vial (Scintillation Vial, KG-33 Borosilicate Glass, VWR International, Radnor, PA, USA) containing 1.7 ml of medium. For BLM treatment medium was completed by 200 mU/ml BLM (Cell Pharm GmbH, Bad Vilbel, Germany). Glasses were closed with Filter Easy Caps 25 cm 2 (ThermoFisher Scientific, Waltham, MA, USA). The vials were put in a roller system (Stuart Rotator SB3, Bibby Scientific, Staffordshire, United Kingdom) rotating at 5 rpm at 37uC and 5% CO 2 . Every 24 h medium was changed. After 72 h of incubation the lung slices were homogenized for Western Blotting or embedded in paraffin for immunostaining as explained above.

Statistical Analysis
The statistical computation was done upon the recommendation of Dresden Institute for Medical Informatics and Biometry (Dresden, Germany). Statistical analysis was performed pretesting for a Gaussian distribution with Shapiro-Wilk-Test followed by an unpaired and two-tailed student t test. In case of declining normal distribution significance was tested by Wilcoxon-Mann-Whitney-Test (U-Test). Results were usually presented as Box-Whiskers-Plots including the minimum, the 25th percentile, the median, the 75th percentile and the maximum of all values. For cell experiments including bleomycin treatment results were presented as mean 6 standard error of mean (SEM) in relation to the control. The whole statistical analysis was done with GraphPad Prism 5.03 software (GraphPad Software, San Diego, CA, USA). Significance was accepted when the p value was below 0.05 (indicated by *). A minimum of three independent experiments was done.

P2rx7 2/2 Knockout Animals Show Reduced Aqp5 mRNA Expression and AQP-5 Protein Levels
In different lung fibrosis models, a loss of AQP-5 from alveolar epithelial cells has been described, thus indicating AQP-5 as an early injury marker in lung [32,33]. The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Immunohistochemical assessment of lungs from wild type and P2rx7 2/2 mice in situ revealed significant differences in AQP-5 immunoreactivity. Decreased levels of AQP-5 protein expression were detected in P2rx7 2/2 mice compared to wild type in 2-3 month old animals ( Figure 1A). In parallel, western blot analysis revealed decreased protein content of AQP-5 in P2rx7 2/2 mice ( Figure 1A). Quantitative real-time RT PCR analysis showed less abundant content of Aqp5 mRNA in the homogenates of lungs from P2rx7 2/2 mice compared to the control group ( Figure 1A).
Quantitative real-time RT PCR and western blot analysis of lungs from 11-12 month old wild type and P2rx7 2/2 mice have shown a slightly increase of AQP-5 in the P2rx7 2/2 mice ( Figure 1B). Immunohistochemical assessment of lungs from these animals in situ showed no increase in AQP-5 immunoreactivity in the P2rx7 2/2 mice ( Figure 1B).

Analysis of Fibrosis-relevant Proteins in P2rx7 2/2 Animals Over a Period of 12 Months
It was shown that chronic lung injury and lung fibrosis is associated with decreased protein and mRNA expression of AQP-5 in the lung [15,33]. Therefore, we have analyzed the P2rx7 2/2 mice over a period of 12 months to determine whether the absence of the P2X7R together with reduced AQP5 protein content is spontaneously leading to the development of fibrosis-like lesions.
We have examined the P2rx7 2/2 mice on alterations similar to changes observed in early stages of lung fibrogenesis: (1) extracellular matrix accumulation, (2) alveolar epithelial changes, particularly AT I cell injury with loss of AT I cell specific antigens, and (3) increased apoptosis of pulmonary cells. We included in this study 2-3 and 11-12 month old animals.
A hallmark of fibrosis is the presence of fibroblastic foci with differentiated fibroblasts which show myofibroblast phenotypes and which secrete in abundance extracellular matrix proteins [34]. Using Sirius Red staining, we found no differences of total collagen in P2rx7 2/2 mice in comparison to the wild type mice regardless of age (Figure 2A). Previously we have shown that immunohistochemical assessments of lungs from wild type and P2rx7 2/2 mice revealed no changes in the protein content for collagen type I [22]. For determination of the expression of collagen1a1 and col-lagen1a2, total RNA from cell homogenates of wild type and P2rx7 2/2 mice were prepared and subjected to quantitative realtime RT PCR. The collagen1a1 and collagen1a2 expression in the homogenates of lungs from 14 wild type and 17 P2rx7 2/2 mice showed no differences in the expression of mRNA (data not shown).
In the normal lung, AT II cells express CD44 epithelial isoforms (CD44v1-10) predominantly at the basolateral site indicating a function as adhesion protein mediating contact to the underlying matrix [35]. CD44 is further involved in uptake and degradation of hyaluronate in lung and lymphoid tissue [36,37]. Advanced stages of disease could show that alveolar epithelial cells in fibrotic foci were either devoid of CD44 epithelial isoforms or overexpressed at places with epithelial hyperplasia [35]. Immunocytochemical detection of CD44v10 expression in paraffin sections of lungs from P2rx7 2/2 and wild type mice revealed no differences in the CD44v10 expression pattern between P2rx7 2/2 and wild type mice regardless of age ( Figure 2B), thus indicating normal AT I and AT II differentiation.
Immunohistochemically detectable differences of apoptosis in lung parenchymal cells were analyzed by detection of active caspase-3. The level of apoptosis in the P2rx7 2/2 mice was comparable with the wild type mice ( Figure 2C).
Caveolin-1 (Cav-1) is an integral membrane protein of AT I and other lung cells. Its relevance in fibrotic diseases has already been recognized in earlier studies [38,39]. The Cav-1 expression in the homogenates of lungs from 2-3 month old P2rx7 2/2 mice showed no differences in comparison to wild type mice. Western blot analysis of lung homogenates showed also no differences in the Cav-1 content in the P2rx7 2/2 mice both in 2-3 month as well as in 11-12 month old animals ( Figure 3A). Immunohistochemical assessment of lungs from wild type and P2rx7 2/2 mice showed no differences in the immunoreactivity for T1a, an AT I cell specific protein and early marker of lung injury in fibrosis [40]. Western blot analysis and quantitative real-time RT PCR of lung homogenates demonstrated also no differences in P2rx7 2/2 and wild type mice regardless of age ( Figure 3B).
Connexins, the elementary units of intercellular gap junctions, are known to be expressed in AT I and II cells and hemichannel activity shows some functional relationship to P2X7R activity [41].
Enhanced immunoreactivity for connexin 43 (Cx43) was detected in epithelial cells at earliest stage during the development of radiation-induced pulmonary fibrosis [30]. Immunohistochemical assessments and western blot analysis from 2-3 month old wild type and P2rx7 2/2 mice showed no significant differences in the amount of Cx43 ( Figure 3C). Quantitative real-time RT PCR analysis from 2-3 month old animals showed no significant differences in the content of Cx43 mRNA in the homogenates of lungs from P2rx7 2/2 mice compared to the control group ( Figure 3C). Western blot analysis from 11-12 month old animals showed also no significant differences in the content of Cx43.
Altogether, all proteins with putative properties as early indicators of fibrosis in alveolar epithelial cells showed no alterations in P2rx7 2/2 mice during the course of age.
Immunohistochemistry of PCLS from Lungs of Wild Type and P2rx7 2/2 Mice Revealed Decreased Expression of AQP-5 in AT I After BLM Treatment Figure 4 illustrates the immunohistochemical assessment of paraffin embedded PCLS prepared from 1-3 month old wild type and P2rx7 2/2 mice before and after BLM exposure. After 3 days of BLM treatment a decrease in AQP-5 protein content was detected in PCLS of lungs of the wild type mice in comparison to the untreated wild type mice ( Figure 4A, C). Interestingly, the P2rx7 2/2 mice treated with BLM showed a stronger reduction in the AQP-5 protein content in comparison to the untreated P2rx7 2/2 mice. To analyze the level of early fibrosis, we assessed also collagen by using conventional Sirius Red staining ( Figure 4B). There were no signs of overproduction of collagen after BLM exposure of PCLS from wild type and knockout animals under the described conditions.

The AQP-5 Expression is P2X7R-dependent
Finally we analyzed whether the stimulation of P2X7R in E10 and MLE-12 cells with BLM induces changes in the AQP-5 protein expression. An increase of the AQP-5 expression was observed after 24 h in both cell lines exposed to BLM ( Figure 5A, B). To test the hypothesis, that the P2X7R is responsible for the increased AQP-5 expression after BLM treatment, we checked the AQP-5 expression after inhibition of the P2X7R by oxATP after 24 h. The resulting western blots demonstrated a decreased amount of AQP-5 after oxATP treatment in comparison to BLM treated cells (Figure 5 A, B).
To study the time-course of AQP-5 and P2X7R protein expression levels, E10 and MLE-12 cells were lysed after 24, 48 and 72 hours of BLM treatment (100 mU/ml) and subjected to Western blot analysis. P2X7R and AQP-5 protein expression are significant increased after 24 h exposure to BLM in both cell lines. Also after 48 and 72 hours the protein content of AQP-5 and P2X7R is increased (Figure 6 A, B).
The time course of AQP-5 of PCLS from wild type mice treated with BLM (200 mU/ml) have shown, however, an increase in AQP-5 after 24 h and a decrease of AQP-5 after 48 and 72 h exposure to BLM (data not shown). A direct comparison of both experimental settings is not possible, since we needed to use lower concentration of BLM in the cell culture model, to prevent the induction of apoptosis in E10 and MLE-12 cells.

Discussion
Our results in the present study indicate that P2rx7 2/2 mice during the course of age did not develop any signs of pulmonary fibrosis or any other signs of lung injury. We could not find any enhancement or alteration in the expression of proteins, which can be regarded as early indicators of fibrosis such as collagen accumulation, loss of T1a [40] increase in CD44v10 or Cx43 [42,43,44]. Moreover, Riteau et al. (2010) [6] showed that the development of a fibrosis is reduced in absence of the P2X7R by demonstrating that the amount of collagen was strongly reduced in BLM treated P2rx7 2/2 mice.
In the present study, we have shown that as a prominent feature AQP-5 is downregulated in the 2-3 month old P2rx7 2/2 animals. This reduction was reversible, moreover AQP-5 expression increased strongly in older animals, where also no fibrotic lesions in the lung parenchyma were detectable. From these findings we suggest that the early loss of AQP-5 described in different fibrosis models [15] is not necessarily connected with the development of this disease but somehow connected with the function of other proteins of AT I cells. In some animals deficient in AT I cellspecific antigens, the lung Aqp5 mRNA is also decreased [10,11]. For example the T1a knockout mice express about 50% of the normal levels of Aqp5 mRNA [10]. It was previously shown that T1a is neither involved in water transport nor regulates AQP-type water channels [45].
As a second outcome of the present study, PCLS of wild type and P2rx7 2/2 mice treated with BLM showed a decrease in the AQP-5 protein content. Remarkably, PCLS of the P2rx7 2/2 mice treated with BLM showed a stronger decrease in the protein expression of AQP-5 compared to untreated P2rx7 2/2 mice. The reason for this decrease is not clear. Treatment of PCLS with bleomycin did also not result in the development of fibrosis-like changes in the lung.
The alveolar epithelium seems to play a central role in gas exchange and regulation of lung water content in response to acute and subacute lung injury. AT I cells exhibit the highest water permeability ever reported for a mammalian cell [46]. The AT I cells likely regulate alveolar fluid volume, composition and clearance. The bulk of water flux across this epithelium depends on the AT I cell specific membrane water channel AQP-5. The airspace-capillary water permeability in AQP-5 knockout mice has a 90% decrease, suggesting the importance of AQP-5 under normal conditions in the lung [1]. Gabazza et al. (2004) [15] demonstrated that chronic lung injury and lung fibrosis is associated with decreased expression of AQP-5 in the lung. In the present study, treatment of immortal lung cell lines with BLM resulted in an increase in AQP-5 protein content. This obvious discrepancy between the PCLS and cell culture data, decreased AQP-5 after BLM exposure on the one side and increased AQP-5 on the other side, tangents a general problem in the comparison of experiments at the organoid level (explant culture) with experiments at the single cell level. We cannot exclude additional indirect effects derived from the absence or presence of other lung cell types in the present experimental settings. Nevertheless, we further demonstrated in the present study that P2X7R activation in alveolar epithelial cells by BLM is implicated in the upregulation of AQP-5 protein expression in vitro. The signal transduction of the P2X7R is supported mainly by the depolarization of the cell membrane and by increasing the intracellular Ca 2+ concentration [47]. CaM is a ubiquitous Ca 2+ sensor in which the binding regulates the activity of various ion channels, and thereby the ion balance of the cell can be maintained [48,49]. Previously we have shown that BLM treated alveolar epithelial cells show an increase in the expression of P2X7R and CaM [22].
CaM is a Ca 2+ -binding protein and a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. Many membrane proteins that act as transporters and channels, such as the cardiac and neuronal voltage-gated ion channels [50,51,52,53], gap junctions [54,55] and the aquaporin water channels [56,57] are modulated by CaM in response to cytoplasmic Ca 2+ fluctuations.
Lipid rafts are known as cholesterol-and glycolipid-enriched microdomains [58] and have been implicated in membrane sorting and trafficking, as well as receptor signalling [58,59,60]. As previously reported, cevimeline induces the translocation of AQP-5 with lipid rafts from the cytoplasm to the apical plasma membrane (APM) by Ca 2+ signalling and the dissociation of AQP-5 from lipid rafts to non-rafts in the APM [61]. It is noteworthy that lipid rafts are implicated in the sorting of some membrane proteins to the APM domain of epithelial cells [62]. Studies using membrane cholesterol depleting reagents, for example statins, have supported the role of membrane rafts in regulation of AQP-2 trafficking [63]. Previously, we have shown that two populations of the P2X7R are detectable in the plasma membrane of E10 cells. A discrete population of P2X7 was associated with lipid rafts [64].
In summary, the results of the present study show, that an activation of P2X7R in vitro is connected with an increase of AQP-5. Otherwise, if P2X7R is inhibited, a down regulation of AQP-5 was observed. These data suggest that P2X7R effects AQP-5 activity directly or indirectly. It may be that CaM is directly involved in these processes. The activation of the P2X7R could lead to the translocation of AQP-5 in lipid rafts. Further studies will be necessary to clarify the relationship between P2X7R activation and a potential localization of AQP-5 in lipid rafts of AT I cells.