Mechanisms of ion regulation and of metabolic control in swimbladder gas gland cells

Gas gland cells of the swimbladder of the European eel Anguilla anguilla can be cultured in Collagen Typ I coated petri dishes (3). Histological examinations of these cells revealed that they are flat and show no obvious polarity, which is in stark contrast to the appearance of gas gland cells in vivo. One major goal of project P11837-BIO was to improve the culture conditions by using permeable supports in order to establish a cell culture model for gas gland cells with histological and functional properties comparable to the in vivo situation. A comparison of various commercially available permeable supports gave the best results with collagen coated Anopore or Whatman Anodisc membranes. The primary cultures were grown until they reached confluence or near confluence and then transferred into a superfusion system, in which both sides of the membrane could be supplied separately with different culture media. In a first series of experiments saline was supplied to the apical membrane, and glucose containing DMEM medium was supplied to the serosal membrane of the cells. Cells grown in a petri dish are almost flat and show no membrane specialization like the basolateral labyrinth, well known from gas gland cells in vivo. After two to four days in the superfusion system, however, cells grown on an Anodisc membrane are cuboidal and histologically show a clear polarity. The apical membrane is characterized by small microvilli, and the basolateral membrane shows a large number of membrane foldings. Thus, cells grown on permeable supports look similar to gas gland cells in vivo. To support this conclusion we localized the Na+ /K+ -ATPase in the membranes of cultured cells by using immunohistochemistry. Incubation of the cells with commercially available fluorescent antibodies against Na+ /K+ - ATPase indeed revealed a concentration of this protein in lateral and in basal membranes, but there was no fluorescence in apical membranes. The presence of surfactant in the swimbladder is well established, but there was no study elucidating where this swimbladder surfactant is produced. The presence of lamellar bodies in cultured gas gland cells as well as exocytotic events visible on electronmicroscopical pictures suggested, that surfactant is produced and released from gas gland cells. From Dr. M. Post from the Hospital for Sick Children in Toronto we obtained fluorescence labeled specific antibodies against surfactant protein A (SP-A). Specificity of these antibodies was tested in a Western blot, and we obtained the typical band at about 65 kD, probably representing the dimer of SP-A, and occasionally a band at 120 kD. There was an additional double band at about 45 kD, which has already been observed in previous studies using SP-A antibodies. 45 kD is the molecular weight of surfactant protein D (SP-D), and a Western blot using specific antibodies against SP-D (obtained from Dr. G. Putz, Institut für Anästhesiologie, Universität Innsbruck) indeed produced a double band at 45 kD. It therefore is quite possible, that the antibody against SP-A shows a cross-reaction with SP-D. The localization of SP-A in culture cells revealed a remarkably polar distribution. SP-A was restricted to the luminal and adjacent lateral membranes, and there was no surfactant protein in the basal membranes. These experiments therefore demonstrated that gas gland cells have a dual function, they produce and release acidic metabolites at the basolateral membranes, and they also produce surfactant, which is released at the luminal membranes. This conclusion was supported by an accompanying study, in which a histological examination of the developing swimbladder of glass-eels revealed the presence of lamellar bodies in gas gland cells and the presence of surfactant in the swimbladder before the initial inflation. Functional polarity of cultured gas gland cells in the superfusion system could be demonstrated by measurement of lactate production and lactate release. About 75% of the lactate was released at the basal side of the cells, only 25% at the apical membranes. Experiments using phenol red as a marker revealed, that the cells growing on permeable supports do not form a perfectly tight epithelial cell layer completely separating the two compartments of the superfusion chamber. It therefore appears quite possible, that 75% lactate release to the basal side is even an underestimate, because some of the lactate diffused from the serosal compartment to the luminal compartment via paracellular pathways along the diffusion gradient. Because swimbladder gas gland cells in vivo are exposed to a gas phase at their luminal membrane, we also tried to culture cells in an air/liquid system. The histology of these cells including the location of SP-A and Na+ /K+ - ATPase is very similar to the one described for the saline/DMEM system. The amount of lactate released to the basal side is consistent with the total amount of lactate released by cells cultured in the saline/DMEM system. This clearly supports the conclusion, that gas gland cells cultured on permeable supports are functionally polar and represent a useful model to study gas gland cell function in vitro. The second major goal was to improve our understanding of gas gland cell function. By histochemical localization of carbonic anhydrase our physiological results from a previous project, showing the presence of a membrane bound carbonic anhydrase facing the extracellular space (2), have been confirmed. Carbonic anhydrase activity was present in the basolateral membranes of the gas gland cells, but not in luminal membranes. In a comparative study swimbladder tissue of the perch was included in the analysis. In contrast to the eel, the perch has a compact gas gland. Nevertheless, using Hanson`s method carbonic anhydrase activity was especially pronounced in gas gland cell membranes near blood vessels. In the membranes of the flat epithelial cells at the swimbladder lumen, however, no carbonic anhydrase activity was found. Carbonic anhydrase apparently plays a pivotal role in gas gland cell function. In contrast to earlier belief, however, no disequilibrium in the CO2/HCO3 - reaction is involved. Carbonic anhydrase activity is present in the cytoplasma as well as in the membranes facing the extracellular space, and this will assure a rapid equilibrium in the CO2/HCO3 - reaction in the cell, but also in the interstitial space. Measurement of proton release at varied extracellular pH demonstrated, that the rate of proton release of gas gland cells is highest at pH 7.4, and reduced at lower as well as at higher pH values. Inhibition of Na+ /K+ -ATPase activity either by incubation with ouabain or removal of extracellular K + reduced the rate of proton release, but it did not abolish proton release completely. Furthermore, inhibition of Na+ /H+ -exchange by 5-(N-methyl-N- isobutyl)-amiloride maximally reduced the rate of proton release by about 60%. This clearly suggests that sodium independent proton release is involved in acid secretion, and a reduction in acid release in the presence of bafilomycin suggests, that a V-ATPase might be involved. Attempts to localize V-ATPase using specific antibodies against the B subunit of V-ATPase of Manduca sexta did not yet give satisfying results, possibly because the binding of these antibodies to the fish protein was not adequate. Using degenerate RT-PCR primers (obtained from Dr. J. Claiborne, Dept. of Biology, Georgia Southern University, Statesboro), we were able to prove that V-ATPase is expressed in gas gland cells by amplification and cloning of a section of the B subunit of the V-ATPase. Using RACE-PCR, we completed this study and could show that two different isoforms of the B subunit are present in gas gland cells, and both isoforms have been cloned and sequenced (GenBank accession numbers AF 099743 and AF179250). In the literature two different isoforms of this B subunit have only been reported for mammals, where a kidney isoform is present in intercalated cells of the kidney, while a brain isoform appears to be present in a large number of different tissues. Our results indicate that the presence of different isoforms serving different physiological functions may not be restricted to mammals. Gas gland cells secret surfactant, and this requires the presence of V-ATPase in the membranes of the lamellar bodies. The acidification of the extracellular space down to pH 6.5 and the reduction of acid secretion in the presence of bafilomycin suggest, that a V-ATPase may also be involved in proton transfer through the plasma membrane. In this situation the presence of two different isoforms of the B subunit of V-ATPase certainly stimulates the idea that these two isoforms serve different functions. Based on the sequence of these two subunits we currently try to identify and localize the two different isoforms in cells and tissue using specific antibodies and in situ hybridization. Using the fluorescent probe BCPCF we also analyzed intracellular pH regulation of gas gland cells. The results of a first series of experiments showed that in the physiological range of pH 7.4 to 7.8 intracellular pH was slightly acidic in relation to the extracellular pH value. Application of amiloride or of specific inhibitors blocking the anion exchange revealed that both, the Na+ /H+ -exchanger and the anion exchanger are involved in the regulation of intracellular pH. Although these pharmacological studies are not yet completed the results obtained so far clearly demonstrate that gas gland cells are equipped with several different mechanisms catalyzing the transfer of acid equivalents through the cell membrane. This redundancy in transport mechanisms probably ensures that gas gland cells can secret acidic metabolites over a wide range of extracellular pH values, and, depending on the activity status of the gas gland cells, the extracellular pH measured in swimbladder blood indeed varies between pH 7.8 and 6.6 (1).