Swimbladder gas gland cells cultured on permeable supports regain their characteristic polarity
Institut für Zoologie und Limnologie, Universität Innsbruck, A-6020 Innsbruck, Austria
*Author for correspondence (e-mail: Bernd.Pelster{at}uibk.ac.at)
Accepted September 24, 2001
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Summary |
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Key words: Swimbladder, gas gland cell, epithelial cell, cell culture, Anguilla anguilla, air/liquid culture.
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Introduction |
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In this situation, the use of cultured cells as a surrogate model has often proved advantageous in enhancing our understanding of the cellular physiology of complex organs and organ systems. Cultured branchial epithelia, for example, have been used to analyze the ion-transport characteristics of freshwater gills (Wood and Pärt, 1997), and studies on cultured kidney tubular cells have significantly enhanced our knowledge about the mechanism of renal acidbase regulation (Brown and Stow, 1995
; Feifel et al., 1997
; Alexander et al., 1999
).
Similarly, the first insight into the mechanisms of ion transport and of metabolic control in gas gland cells was obtained using cultured cells. Working with isolated and cultured cells, it could be shown that gas gland cells secrete acid via the Na+/H+ exchanger, Na+-dependent anion exchange and diffusion of CO2 (Pelster, 1995; Pelster and Niederstätter, 1997
). In addition, the presence of a V-ATPase was demonstrated, and this may also contribute to the secretion of protons generated in the glycolytic pathway (Niederstätter and Pelster, 2000
).
Initially acid production and secretion were considered to be the most important function of these cells, but a recent study has revealed that gas gland cells are also responsible for the production and secretion of surfactant into the swimbladder lumen (Prem et al., 2000). Consequently, it is now clear that gas gland cells produce and secrete acid at their basolateral membranes, and surfactant at their apical membranes. Thus, a cell culture system in which gas gland cells retain their polarity would be a useful model for studying the mechanisms of ion regulation and ion secretion. Several studies have shown that the polarity of fish epithelial cells can be retained in culture by using permeable supports (Dickman and Renfro, 1986
; Wood and Pärt, 1997
). The goal of the present study, therefore, was to establish a primary culture system for gas gland cells in which the cells retain their morphological and physiological polarity. Physiological polarity was assessed by measuring lactate release. Histological polarity was evaluated by electron microscopy and by immunohistochemical localization of Na+/K+-ATPase.
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Materials and methods |
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The superfusion system was custom-designed as described (Prem and Pelster, 2000). Briefly, the Perspex superfusion chambers were supplied with cell culture medium and saline (see above) by an eight-channel peristaltic pump (Ismatec IPC-8, Wertheim-Mondfeld, Germany). The flow was adjusted to a constant rate of 1 ml h1. The fluid leaving the apical and basal superfusion chambers was collected as 1 ml samples in Eppendorf tubes placed in a custom-made fraction collector. The fraction collector allowed the samples to be cooled for a few hours until they were frozen for further analysis at 80°C. The whole arrangement was autoclaved prior to each run to avoid any bacterial contamination.
Occasionally, cell preparations showed rather poor adherence and the cell density on the filter membrane was low. To check the cell density in the superfusion system, Phenol Red was added to the fluid supplied to the basal membranes (Jovov et al., 1991), and only preparations in which the Phenol Red leakage from the basal to the apical chamber was less than 2.5 % were used for experiments. In these preparations, the cell density on the filter membrane was approximately 400,000 cells cm2. In the air/liquid culture system, the fluid supplied to the apical side of the superfusion system was replaced by humidified air. Humidification of the air was achieved by bubbling the air through a series of water bottles.
Physiological measurements
The lactate content of the medium used for the superfusion system was determined in an enzymatic test according to the principle described by Bergmeyer (1974). Measurements were performed in a plate reader (fmax, Molecular Devices, Munich, Germany) using the difference in the fluorescence signals of NAD+ and NADH.
The transepithelial resistance of cells cultured on Costar Transwell 13 membranes was determined using an EVOM epithelial voltohmmeter with ENDOHM 12 electrodes (World Precision Instruments, Berlin, Germany). In these experiments, cells were supplied with DMEM F12 on the basal side, and with saline on the apical side. Media were exchanged daily. Control measurements were performed using exactly the same system, but no cells were seeded on its filter membranes. Measurement of the transepithelial resistance within the superfusion system proved impossible.
SDS-PAGE and western blot analysis
Protein from a swimbladder homogenate was separated by sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) using the NuPage buffer system. Electrophoresis was performed with Power Ease 500, X-Cell II using NuPage 10 % Bis Tris gels (all from Novex, Germany). The SDS-PAGE was performed under reducing conditions using dithiothreitol (DTT), at 125 µmol l1.
Electrophoretic transfer of proteins to a nitrocellulose membrane was performed using Power Ease 500 (Novex, Germany). The transfer was conducted for 1 h at a constant voltage of 25 V (160 mA). The nitrocellulose membranes were placed in a sealed bag containing 10 % BSA, 10 % fetal calf serum (FCS) and 0.1 % Tween 20 (Sigma) in 100 mmol l1 phosphate buffer and gently agitated for 1.5 h at room temperature (2224°C). After washing, the membranes were incubated overnight at 4°C with a chicken Na+/K+-ATPase antibody (Biogenesis, Poole, Great Britain) diluted 1:50,000 (v/v) in phosphate buffer containing 1 % BSA, 1 % FCS and 0.1 % Tween 20. The membranes then were washed and incubated for 1 h with Sigma anti-chicken IgG (A9046) at 1:10,000 (v/v) conjugated with horseradish peroxidase, in phosphate buffer with 1 % BSA, 1 % FCS and 0.1 % Tween 20 at room temperature. Antibody binding was visualized by enhanced chemiluminescence (ECL; Amersham Life Science).
Immunohistochemistry
Cells were fixed in 4 % paraformaldehyde in 10 mmol l1 phosphate buffer (pH 7.4) for 1 h, washed, blocked for 1 h with 0.2 % I-Block (Tropix, USA) and 0.2 % Triton X-100 (Sigma) in 10 mmol l1 phosphate buffer and incubated with the chicken Na+/K+-ATPase antibody (Biogenesis, Poole, Great Britain) at a dilution of 1:100 in blocking buffer overnight at 4 °C. After washing, the samples were incubated for 1 h with Sigma anti-chicken IgG (A9046) fluorescein isothiocyanate (FITC)-conjugated antibody (Dako), diluted 1:100 in blocking buffer, and embedded in Vectashield (Vector Laboratories). Analysis was performed with a laser-scanning microscope (Zeiss, LSM 510). Cells were sectioned with a vertical resolution of 0.3 µm.
Electron microscopy
Cells on permeable supports were placed into 2.5 % glutaraldehyde in 10 mmol l1 phosphate buffer (Dulbeccos formula, pH 7.4) for 30 min, washed and postfixed in 2 % osmium tetroxide containing 2.5 % potassium ferrocyanide for 90 min at 4°C. The samples were dehydrated through a graded acetone series and embedded in Spurrs 15 low viscosity resin (Spurr, 1969). Ultrathin sections were cut with an Ultracut E (Reichert, Austria), double-stained with uranyl acetate and lead citrate and examined in an EM902 (Zeiss, Germany).
Data analysis
Data obtained with the laser-scanning microscope were processed on an O2 workstation (Silicon graphics) using an appropriate software package (Imaris 2.6.8) from Bitplane (Bitplane AG). For contrast enhancement and deconvolution, the software package Huygens, 2.0 (Scientific Volume Imaging BV, Netherlands) was used. Three-dimensional reconstruction of the cells was achieved with the Isosurface module of Imaris 2.6.8.
Statistically significant differences in the observations were evaluated using a one-way analysis of variance (ANOVA) followed by a multiple-comparison procedure (Bonferoni). Significance was accepted when P<0.05. Data are presented as means ± standard error of the mean (S.E.M.). N represents the number of filters from 35 different fish.
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Results |
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Using immunohistochemistry Na+/K+-ATPase was found to be largely confined to the lateral membranes, and was not present in apical membranes (Fig. 3). Fig. 4 shows a three-dimensional reconstruction of a cell obtained after deconvolution of the data. Fluorescence is found in the lateral membranes and in the basal parts of the cultured cell. Membrane foldings penetrate the basal part of the cells (see Fig. 1), and the fluorescence islets observed inside the cell represent these membrane foldings. The specificity of the commercially available antibody was tested using western blot analysis. Bands with a molecular mass of approximately 40 kDa and 50 kDa were observed in all homogenates tested (Fig. 5). In addition, a band with a molecular mass of approximately 60 kDa was present.
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Discussion |
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In vivo, eel gas gland cells are exposed to a gas phase on their apical side, and we have successfully developed an air/liquid culture system to mimic the in vivo situation as closely as possible. An air/liquid culturing system has been used for mammalian lung epithelial cells (Dobbs et al., 1997) and, in this situation, surfactant-secreting type II epithelial cells have been cultured successfully.
Measurement of the electrical resistance of cultured gas gland cells revealed that the epithelia have only a low resistance. Gill cells in culture form epithelial layers with an electrical resistance of several thousand cm2, which is 3050 times higher than the resistance measured for gas gland cells in the present study. In cultured freshwater gill cells, the magnitude of the transepithelial resistance increases with decreasing salinity of the media on the apical side (Wood and Pärt, 1997
; Avella and Ehrenfeld, 1997
; Gilmour et al., 1998
). Gill cell epithelia from seawater fish in turn have a high resistance (Avella and Ehrenfeld, 1997
), which clearly demonstrates that the resistance of these preparations is significantly modified by the composition of the medium. In contrast to gill cells, which form a barrier to the low-osmolarity external medium in freshwater trout, the apical side of gas gland epithelial cells is facing a gas phase. The apical surface of gas gland cells is covered with hydrophobic surfactant (Prem et al., 2000
), so that a very tight epithelium is not necessary. This may explain the low electrical resistance. For native trachea sheets studied in an Ussing chamber, resistance values of approximately 125150
cm2 have been reported (Yamaya et al., 1992
), which is within the range of values measured for our cultured gas gland cells after 5 days in culture.
The physiological function of gas gland cells is to produce lactic acid and CO2, which are secreted at their basolateral membranes (Pelster et al., 1989), and to produce surfactant, which is released at their apical membranes (Prem et al., 2000
). Cultured gas gland cells produce and release lactic acid, and a comparison of the lactate contents in the superfusate of the apical side with that of the basal side revealed that approximately 6070 % of the lactate is released at the basal membranes. This generates a gradient in lactate concentration from the basal chamber of the perfusion system to the apical side, which favours lactate diffusion from the basal to the apical chamber. Measurements of electrical resistance demonstrated that gas gland cells form a leaky epithelium. It may well be, therefore, that the measured ratio of lactate release to the apical and the basal sides is an underestimate because of paracellular diffusion of lactate from the basal chamber of the superfusion system to the apical side.
In the air/liquid culture, all the lactate was released to the basal side, and there was no fluid transfer to the apical side. At first glance, this may contradict the idea of lactate diffusion between the two sides of the Ussing chamber. However, assessing the tightness of the epithelial layer using Phenol Red clearly demonstrated that some leakage may occur in the liquid culture system, and we used only epithelial layers in which this leakage rate was below 2.5 %. Lactate is a much smaller molecule than Phenol Red, and if Phenol Red penetrates the cell layer, lactate should penetrate as well. In the air/liquid system, however, surfactant has to be taken into account. While in the liquid culture system, surfactant is secreted into a liquid phase and may be washed away; in the air/liquid culture system, the hydrophobic surfactant will cover the apical surface of the cells and thus block paracellular fluid transfer.
It is interesting to note that in the air/liquid culture system the amount of lactate released by the cells significantly exceeds that of cells in the liquid culture system. Previous experiments have shown that the rate of acid secretion as well as the rate of lactate production and release by the swimbladder tissue of the European eel decrease under hypoxic conditions (Pelster and Scheid, 1992, 1993
). This observation could be explained, in part, by a decrease in blood supply and thus in glucose supply to the swimbladder tissue during hypoxia. The observed decrease in metabolic activity, however, far exceeded the decrease in blood supply, so these experiments provided clear evidence for a downregulation of metabolic activity of the swimbladder tissue during hypoxia (Pelster and Scheid, 1993
). Given the significantly higher oxygen capacity of air compared with saline, the oxygen supply to the cells in the air/liquid system is certainly much better than in the liquid culture system. Thus, the higher rate of lactate release in the air/liquid experiments may be explained by a better oxygenation of the cells. This would imply that the results of the present study support the hypothesis that the metabolism of gas gland cells is, in part, controlled by the level of oxygen availability. In this context, it should be mentioned that gas gland cells lack a Pasteur effect (DAoust, 1970
). Thus, in gas gland cells, glycolytic flux does not decrease at higher oxygen tensions. This certainly makes sense given that gas gland cells are typically exposed to hyperoxic conditions (Pelster, 1997
) and, to ensure continued gas secretion, must continue to produce lactic acid in the glycolytic pathway. The explanation for this unusual effect of oxygen on the metabolic activity of gas gland cells must await further experimentation.
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Acknowledgments |
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