Gas Gland Cells Research Articles

Development of the swimbladder in the European eel ( Anguilla anguilla).

The swimbladder of the adult eel, Anguilla anguilla, with its bipolar countercurrent system, the rete mirabile, is a widely used model for swimbladder function, but very little is known about the development of this swimbladder. Our histological studies on the developing swimbladder revealed that during metamorphosis the swimbladder becomes present as a dorsal outgrowth of the esophagus. It is filled with surfactant, and gas was not detected in the swimbladder. In the young glass-eel, the epithelial (gas gland) cells of the swimbladder are columnar, but do not yet have the typical basolateral labyrinth established in adult animals. Few blood vessels are found in the swimbladder tissue, and the submucosa is present as a thick layer of connective tissue, giving a large diffusion distance between blood vessel and swimbladder lumen. Within the next 2 or 3 months of development, gas gland cells develop their typical basolateral labyrinth, and the thickness of the submucosa is significantly reduced, resulting in a short diffusion distance between blood vessels and the swimbladder lumen. The first filling of the swimbladder with gas is observed while the gas gland cells are still in a poorly differentiated status and it appears unlikely that these cells can accomplish their typical role in gas deposition. The presence of small gas bubbles in the swimbladder as well as in the ductus pneumaticus at the time of initial swimbladder inflation suggests that the swimbladder is filled by air gulping or possibly by taking up gas bubbles from the water.

Swimbladder gas gland cells cultured on permeable supports regain their characteristic polarity.

A cell culture system has been developed in which swimbladder gas gland cells from the European eel (Anguilla anguilla) were cultured on a permeable support. Cells seeded on Anodisc 13 (Whatman) or Costar Transwell 13 mm membranes form a confluent cell layer within the first 2 or 3 days of culture but, on the basis of measurements of transepithelial resistance, it is a "leaky" cell layer. In a superfusion system, the apical and basal sides of the cells were superfused asymmetrically, with saline on the apical side and a glucose-containing cell culture medium on the basal side. Under these conditions, the cells continuously produced lactic acid, and approximately 60-70 % of this lactate was released at the basal side. To mimic the in vivo situation, the saline solution supplied to the apical side was replaced by humidified air in an additional series of experiments. Cells cultured in an air/liquid system produced even more lactate, and this lactate was only released to the basal side; there was no leakage of fluid to the apical side. After 4 or 5 days in the superfusion system, the cells were fixed for histological examination. The cells were columnar, similar to gas gland cells in vivo, and showed a clear polarity, with some small microvilli at the apical membrane and extensive membrane foldings at lateral and basal membranes. Immunohistochemical localization of Na+/K+ -ATPase revealed that this ATPase was present mainly in the lateral membranes; it was never found in the apical membranes. Cells cultured in the air/liquid system showed a similar structure and polarity.

The generation of hyperbaric oxygen tensions in fish.

Surprising inventiveness in the molecular interactions in fish hemoglobins that express the Root effect (decreased oxygen-carrying capacity at low pH) and in metabolic adaptations in swim bladder gas gland cells and retinal tissues causes local acidification of blood and generates hyperbaric oxygen tensions that drive oxygen into the swim bladder (regulating buoyancy) and ensures the oxygen supply to the avascularized retinae.

Swim bladder gas gland cells produce surfactant: in vivo and in culture.

Electron microscopical examination of gas gland cells of the physostome European eel (Anguilla anguilla) and of the physoclist perch (Perca fluviatilis) revealed the presence of significant numbers of lamellar bodies, which are known to be involved in surfactant secretion. In the perch, in which the gas gland is a compact structure and gas gland cells are connected to the swim bladder lumen via small canals, lamellar bodies were also found in flattened cells forming the swim bladder epithelium. Flat epithelial cells are absent in the eel swim bladder, in which the whole epithelium consists of cuboidal gas gland cells. In both species, Western blot analysis using specific antibodies to human surfactant protein A (SP-A) showed a cross-reaction with swim bladder tissue homogenate proteins of approximately 65 kDa and in the eel occasionally of approximately 120 kDa, probably representing SP-A-like proteins in a dimeric and a tetrameric state. An additional band was observed at approximately 45 kDa. Western blots using antibodies to rat SP-D again resulted in a single band at approximately 45 kDa in both species, suggesting that there might be a cross-reaction of the antibody to human SP-A with an SP-D-like protein of the swim bladder tissue. To localize the surfactant protein, eel gas gland cells were cultured on permeable supports. Under these conditions, the gas gland cells regain their characteristic polarity. Electron microscopy confirmed the presence of lamellar bodies in cultured cells, and occasionally, exocytotic events were observed. Immunohistochemical staining using an antibody to human SP-A demonstrated the presence of surfactant protein only in luminal membranes and in adjacent lateral membranes. Only occasionally, evidence was found for the presence of surfactant protein in lamellar bodies.

Infection of the glass-eel swimbladder with the nematode Anguillicola crassus.

The ability of the nematode Anguillicola crassus to infect eel larvae (glass-eel stage) was tested. The results show that glass-eels fed on infected copepods, the natural intermediate host of the nematode, can be infected. Light microscopical examination of the infected developing swimbladder tissue revealed that the infection results in a significant thickening of the connective tissue. The basolateral labyrinth of gas gland cells is very much reduced in infected swimbladders, and the distance of gas gland cells to blood capillaries is enlarged. Critical swimming speed, defined as the speed where the larvae were no longer able to swim against the current, was similar in infected and uninfected animals. At intermediate speeds (about 60-80% of critical swimming speed) infected eels showed a slightly higher swimming activity than control animals. Resting oxygen consumption, measured as an index of metabolic activity, within the first 2 months of infection was higher in control animals, which may be due to a reduced rate of activity in infected glass-eels. By 4-5 months after the infection, however, it was significantly higher in infected animals. This may indicate that at this stage a higher activity of the animals is required to compensate for the increase in body density, but swimming performance of infected and non-infected glass-eels was not significantly different. Oxygen consumption during swimming activity, measured in a swim tunnel at 50% of maximal swimming speed, also was not affected. The results thus show that even glass-eels can be infected with A. crassus, and this probably contributes to the rapid spread of the nematode in Europe. While aerobic metabolism during swimming activity is not affected at this stage of infection, the swimbladder tissue shows severe histological changes, which most likely will impair swimbladder function.

Functional morphology of the gas-gland cells of the air-bladder of Oreochromis alcalicus grahami (Teleostei: Cichlidae): an ultrastructural study on a fish adapted to a severe, highly alkaline environment

Oreochromis alcalicus grahami is a small cichlid fish that lives in shallow peripheral lagoons of Lake Magadi, Kenya. The internal surface of the air-bladder is highly vascularized, illustrating possible utilization as an accessory respiratory organ. The wall of the bladder consists of five distinct tissue layers. From the outer to the inner surfaces are: a squamous, undifferentiated epithelial cell; a collagen-elastic tissue space; a smooth muscle tissue band; an inner connective tissue space; and columnar gas-gland cells projecting into the lumen. The cell membrane over the perikarya of the gas-gland cells was sporadically broken. The disruptions were ascribed to possible physical damage by discharging gas-bubbles. Juxtaluminally, the gas-gland cells attached across tight junctions. The cells have highly euchromatic nuclei and conspicuously large intracytoplasmic secretory bodies. On the blood capillary facing (basal) aspect, the cell membrane of the gas-gland cells is highly amplified. The cells insert onto a smooth muscle tissue band. The morphological features and the topographical arrangement of the gas-gland cells in O. a. grahami are indicative of an operative exchange of materials between them and the underlying tissue components especially the blood capillaries. For a fish that subsists in hot, highly saline and alkaline water heavily invested by avian predators and where the partial pressure of oxygen diurnally shifts from virtual anoxia to hyperoxia, development of a versatile air-bladder for efficient buoyancy control conforms to the functional demands placed on it by a unique environment. Illustratively, instead of the gas-gland morphology in O. a. grahami resembling that in the freshwater fishes, the group from which the fish has evolved, it compares more closely to that of the marine fish. This similarity suggests amazing parallel evolutionary adaptation to biophysically corresponding aquatic milieus.

Expression of two vacuolar-type ATPase B subunit isoforms in swimbladder gas gland cells of the European eel: nucleotide sequences and deduced amino acid sequences

The poly(A) + RNA of swimbladder gas gland cells of the European eel Anguilla anguilla was isolated and used for cDNA synthesis. Using a pair of degenerate PCR primers directed towards the evolutionary highly conserved central part of the B subunit of vacuolar type H +-ATPase (V-ATPase) a fragment of 388 bp was amplified. By sequencing the cloned PCR products two different amplicons with a sequence identity of about 86% were obtained. BLASTN searches revealed a high degree of similarity of both to V-ATPase B subunits of other species. The sequences were completed by performing rapid amplification of cDNA ends PCR, subsequent cloning, and sequencing of the obtained products. The expression of two different isoforms of the V-ATPase B subunit is already demonstrated for Homo sapiens and Bos taurus. This is the first report that attributes the same phenomenon to a non-mammalian species, A. anguilla. The first isoform found in eel (vatB2) shows the highest degree of amino acid sequence homology with the human brain isoform (98.2%), the second one (vatB1) with the B subunit sequence of rainbow trout ( Oncorhynchus mykiss) gill and kidney (98,6%). The alignment of the deduced amino acid sequences of vatB1 and vatB2 shows that the highest sequence variation between these two isoforms is found at the amino-terminus, where vatB1 is nine amino acids shorter than vatB2, while at the carboxy-terminus it is two amino acids longer than vatB2. This has also been reported for the human and bovine kidney isoforms when compared with the brain isoforms. Northern blot analysis using specific hybridization probes revealed the expression of two mRNA’s with lengths of about 2.9 kb and 3.5 kb for vatB1 and vatB2, respectively. For mammals, it is well known that V-ATPases containing the kidney isoforms of the B subunit are responsible for the extrusion of protons across the plasma membranes of several cell types. The fact that eel vatB1 seems to share structural features with the kidney isoforms in mammals supports the hypothesis that in gas gland cells a V-ATPase contributes to the acidification of the blood in the swimbladder.

Swimbladder gas gland cells of the European eel cultured in a superfusion system.

Swimbladder gas gland cells are polar epithelial cells which release acidic metabolites through the membranes of an extensive basolateral labyrinth, and secret surfactant via exocytosis at apical membranes. We have developed a method to establish primary cell cultures of gas gland cells in order to establish a model system for physiological analysis of gas gland cell function in vitro. Isolated gas gland cells attach to collagen S coated surfaces. Cells cultured in collagen S coated petri dishes were flat and showed no histological polarity. Cells cultured on Anodisc membranes in a superfusion system, in which the apical and basal side of the cells was supplied with a saline solution and with glucose containing DMEM cell culture medium, respectively, showed a clear polarity similar to the in vivo situation. Measurement of lactate release at the apical side and at the basal side revealed that these cells were functionally polar and secreted at least 70% of the lactate at their basal membranes. Gas gland cells could also be cultured in an air/liquid system, in which the apical membrane was exposed to humidified air. Cells cultured under these conditions released lactate only on the basal side and histologically were similar to cells cultured in the superfusion system.

Acid release in cultured gas gland cells

Acid release in cultured gas gland cells

Gas gland cells regain their characteristic polarity and morphology: a comparison of different culture conditions

Gas gland cells regain their characteristic polarity and morphology: a comparison of different culture conditions

PH-dependent proton secretion in cultured swim bladder gas gland cells.

The pH dependence of acid production and of acid release has been analyzed in cultured gas gland cells of the European eel using a cytosensor microphysiometer. Total acid release of gas gland cells showed an optimum at pH 7.4-7.6, with only a minor reduction at acidic (pH 7.0) as well as at alkaline pH (pH 8.0). The acid production was largely dependent on the availability of extracellular glucose and was almost completely abolished if glucose was replaced by succinate, alanine, or even pyruvate. Phloretin, an inhibitor of glucose uptake, significantly reduced acid release of gas gland cells with a Ki of approximately 1 x 10(-5) M, irrespective of pH. Although the glucose dependence of acid production was not modified by pH, acid release became increasingly sodium dependent with decreasing pH, but at low pH significantly higher sodium concentrations were necessary to achieve maximal rate of proton secretion. This sodium-dependent proton secretion could only in part be inhibited by application of 5-(N-methyl-N-isobutyl)-amiloride. Removal of extracellular potassium caused a slow reduction in the rate of acid secretion. A similar reduction was observed in the presence of ouabain, a specific inhibitor of Na(+)-K(+)-adenosinetriphosphatase, and both effects significantly increased with decreasing pH. The results demonstrate a significant pH dependence of the mechanisms of acid release in swim bladder gas gland cells and indicate that sodium-dependent pathways become especially important at low pH.

Control of acid secretion in cultured gas gland cells of the European eel Anguilla anguilla.

Single cells and cell clusters isolated from the swimbladder epithelium of the European eel Anguilla anguilla attached to collagen S-coated petri dishes and proliferated in a modified Dulbecco's modified Eagle's medium, supplemented with 0.5% fetal calf serum. At a temperature of 20-22 degrees C, the growing colonies reached confluence typically within 6-8 days. Activities of glycolytic and pentose phosphate shunt enzymes remained stable or increased only slightly during the first 10 days of primary culture. Incubated in a defined medium providing glucose as a fuel, gas gland cells in primary culture produced and released lactic acid. The rate of acid secretion of cultured gas gland cells measured with a cytosensor microphysiometer was not influenced by cholinergic stimulation. Similarly, the Ca2+ ionophore A-23187 had no effect. Adrenergic stimulation with epinephrine or the beta-agonist isoproterenol also did not increase the rate of acid secretion, indicating that in gas gland cells the metabolic activity cannot be stimulated via beta-adrenergic stimulation followed by an increase in adenosine 3',5'-cyclic monophosphate (cAMP). Artificially increasing the intracellular concentration of cAMP by incubation with forskolin or the cAMP analogue 8-(4-chlorophenylthio)-cAMP even resulted in a marked reduction in the rate of acid secretion. The results demonstrate that primary cell culture provides a useful means for the analysis of metabolic control and of ion transfer processes in swimbladder gas gland cells.

Mechanisms of acid release in isolated gas gland cells of the European eel Anguilla anguilla

Mechanisms of acid production and of acid release have been analyzed in isolated gas gland cells of the eel swimbladder using a cytosensor microphysiometer. Incubation of isolated cells with oxamic acid caused a dose-dependent decrease in the rate of proton release. At the highest oxamic acid concentration used (20 mmol/l), proton release was reduced by approximately 40%; incubation with sodium fluoride (10 mmol/l) or removal of glucose from the extracellular medium caused 60 and 80% reduction, respectively. NaCN had little effect on proton secretion. Proton release of isolated gas gland cells was largely dependent on the extracellular sodium concentration, and this sodium effect was in part inhibitable by amiloride. A 15-20% reduction in the rate of proton secretion was observed in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of anion exchange. Inhibition of mammalian H(+)-K(+)-adenosinetriphosphatase with omeprazole had no effect, whereas bafilomycin, an inhibitor of vesicular H(+)-adenosinetriphosphatase, induced a 25% reduction in proton secretion. Ethoxzolamide, a membrane-permeable inhibitor of carbonic anhydrase, caused a 60% reduction in proton secretion (inhibition constant = 54.4 nmol/l). Prontosil-dextran, a membrane-impermeable sulfonamide, also reduced the proton release, thus indicating the presence of a membrane-bound carbonic anhydrase facing the extracellular space.

Lactate Production in Isolated Swim Bladder Tissue of the European Eel Anguilla anguilla

Lactate formation of isolated swim bladder epithelium (gas gland cells) of the European eel Anguilla anguilla has been analyzed with a modified Ussing chamber over periods of up to 6 h. The rate of lactate production in these preparations was in the range of 120-250 nmol · min⁻¹ · g⁻¹ wet wt, which is typical for inactive swim bladder tissue, that is, during periods of no gas deposition. The swim bladder epithelium proved to be highly polar; about 80% of the lactate was released to the serosal side and only 20% to the luminal side. Under control conditions the rate of lactate release of the swim bladder epithelium decreased by about 25%-30% over the 6-h incubation period. Neither adrenaline nor the c-AMP analog dibutyryl-c-AMP had any influence on the rate of lactate formation of the tissue. The rate of lactate formation was, however, dependent on the extracellular glucose concentration. A long-lasting stimulation of lactate formation was achieved with bovine insulin and with carbachol at a concentration ...

The Influence of Gas Gland Metabolism and Blood Flow on Gas Deposition into the Swimbladder of the European Eel Anguilla Anguilla

ABSTRACT The effects of blood flow through, and metabolic activity in, the swimbladder epithelium on gas deposition into the swimbladder have been analysed in the European eel, Anguilla anguilla. Blood flow in the artery supplying the retia was measured by Doppler flow probes; measurement of O2 and CO2 content in arterial and venous blood samples from the swimbladder allowed calculation of the rates of O2 removal from, and CO2 addition to, swimbladder blood. 83% of the O2 removed from the blood was transferred into the swimbladder lumen and only 17% was metabolized in the tissue. In spite of the deposition of CO2 into the swimbladder lumen, the CO2 content in rete venous blood was higher than that in arterial blood, indicating production of CO2 in the swimbladder tissue. The respiratory exchange ratio, calculated from O2 consumption and CO2 production of the swimbladder tissue, was significantly greater than one. Gas deposition into the swimbladder increased with increasing swimbladder arteriovenous pH difference, indicating acid release from gas gland cells, and thus their metabolic activity. The rate of gas deposition into the swimbladder increased with increasing blood perfusion of the swimbladder tissue. Under hypoxic conditions, gas deposition was significantly reduced, as was blood flow through the swimbladder tissue. The decrease in gas deposition during hypoxia coincided with a reduction in the swimbladder arteriovenous pH difference. The results therefore demonstrate that the rate of gas deposition is dependent on blood perfusion of the swimbladder tissue and on metabolic activity of the swimbladder tissue, both of which are reduced under hypoxic conditions.

Countercurrent Concentration and Gas Secretion in the Fish Swim Bladder

Many fish possess a gas-filled swim bladder, and the gas molecules enter the bladder by diffusion from its capillaries and epithelium. The high gas partial pressures necessary to drive diffusion are produced in two steps. First, in the blood perfusing the swim bladder capillaries the physical solubility of gases is reduced via the salting-out effect, and gases are released from a chemical binding site via blood acidification (e.g., Root effect for O₂; conversion of HCO₃⁻ to CO₂). These effects result in an increase in the gas partial pressure in blood. In a second step, this initial increase in partial pressure (the "single concentrating effect") is multiplied by back diffusion of gas molecules in the countercurrent system of the rete mirabile. For inert gases, the salting-out effect, induced by the release of lactic acid from the gas gland cells in the swim bladder tissue, results in a probably small reduction of solubility, while the acid-induced decrease in hemoglobin O₂-carrying capacity (Root effect) and the release of CO₂ from HCO₃ ⁻, combined with the release of anaerobically produced CO₂ from gas gland cells, result in a significant single concentrating effect for O₂ and CO₂. The relatively high permeability of the rete capillaries for gases allows for back diffusion, and thus for countercurrent concentration, of gases. The rete mirabile is, however, permeable not only for gases but also for metabolites and salt molecules, and the countercurrent concentration of solutes enhances the countercurrent concentration of gas molecules. This is particularly important for inert gases because their single concentrating effect is small. Back diffusion of CO₂ in the rete mirabile not only results in a countercurrent concentration of CO₂ but also enhances the partial pressure increase of O₂ by decreasing the hemoglobin O₂-carrying capacity within the arterial capillaries of the rete. This interdependence of O₂ and CO₂ may apply to countercurrent systems in other tissues as well, for example, to the skeletal muscle. Hence, the swim bladder rete mirabile may serve as a model tissue for studies of gas exchange and countercurrent blood flow in other organs. Furthermore, the regulation of countercurrent eficiency and gas secretion promises to be an interesting area of research, including also the regulation of metabolic activity and acid production in the gas gland cells.

Ultrastructure and innervation of the swimbladder of Tetractenos glaber (Tetraodontidae).

The general structure, ultrastructure and innervation of the swimbladder of the smooth toadfish, Tetractenos glaber, were examined with light-microscopic, fluorescence-histochemical, and transmission electron-microscopic techniques. The structure of the swimbladder is similar to that of other euphysoclists. Fluorescence histochemistry showed adrenergic fibres in both the secretory and resorptive areas of the swimbladder. Transmission electron microscopy revealed two morphologically distinct axon profiles: type-I profiles containing many small, flattened vesicles; type-II profiles containing both large, granular vesicles and rounded, small clear vesicles in varying proportions. The gas-gland cells and surrounding muscularis mucosae are innervated by both type-I and type-II fibres. Type-I fibres also innervate pre-rete arteries. The rete- and gas-gland capillaries do not appear to be innervated. Arteries running to the resorptive area are innervated by type-I fibres. Both type-I and type-II profiles make contact with the muscularis mucosae in the resorptive area. Only type-I fibres innervate the radial dilator muscle in the oval sphincter region, whereas only type II fibres innervate the circular muscle of the oval sphincter. Type-I fibres took up alpha-methyl-noradrenaline, and could not be found after pre-treatment with 6-hydroxydopamine. They are, therefore, assumed to be adrenergic. Type-II fibres were tentatively identified, by exclusion, as cholinergic.

A Histochemical Study of the Gas Gland Innervation in the Atlantic God,Gadus morhua

A histochemical study was made of the distribution of catecholamines and cholinesterases in two autonomic ganglia closely associated with the swimbladder of the Atlantic cod, Gadus morhua. The “swimbladder nerve ganglion” comprised large (40 μm) neurones, the majority of which are positive for both catecholamines and acetylcholinesterase. It is argued that these neurones are mainly adrenergic postganglionic elements of sympathetic pathways which pass through the vago-sympathetic trunk. The “gas gland ganglion” comprised small (20 μm) neurones, positive for acetylcholinesterase but showing no catecholamine reaction. It is argued that these neurones are cholinergic postganglionic elements of the parasympathetic vagal innervation of gas gland cells.

The ultrastructure of the swimbladder of the toadfish, opsanus tau L.

The anterior chamber of the swimbladder of the toadfish Opsarmustau L. is lined by a single layer of columnar gas gland cells, cuboidal cells that resemble gas gland cells but are located outside of the gas gland region, and squamous cells. Multilamellar bodies are numerous in the gas gland cells and the cuboidal cells and are present in smaller numbers in the squamous cells. Capillaries lie in the lamina propria directly below the epithelial lining. A thick continuous muscularis mucosae and a submucosa consisting of tightly packed cells, cell processes, and connective tissue may contribute to the impermeability to gases of the wall of the anterior chamber. The posterior chamber of the swimbladder is lined by a single type of squamous epithelial cell. Multilamellar bodies were occasionally observed in these cells also. Other types of cells frequently form a partial second layer between the epithelial lining and the basement lamina. A thin muscularis mucosae lies directly below the basement lamina and the capillaries of the posterior chamber are located in the submucosa. The tunica externa is a layer of dense connective tissue that surrounds both the anterior and posterior chambers. Collagen fibrils in the form of tactoids are present in this layer.

The gas bladder of Argentina silus L., with special reference to the ultrastructure of the gas gland cells and the countercurrent vascular bundles.

The orifice between the two chambers of the gas bladder in Argentina silus is surrounded by a sphincter muscle. Gas analyses of the gas bladder contents of fish from 400 meters depth give 0–1% carbon dioxide and 9–72% oxygen. Micro-retia mirabilia form a countercurrent vascular system, and the arterial component has peripherally a sphincter mechanism. The function of the “glandular layer” of the anterior chamber remains uncertain, but the structure indicates secretion into blood capillaries. The lining epithelium of the anterior chamber may secrete some substance into blood or directly into the lumen, which may be involved in a secretory mechanism. This conclusion is not supported by our histochemical tests. The posterior chamber has no micro-retia and the blood vessels have a different origin from those of the anterior chamber. The blood vessels form a plexus of capillaries or sinuses in contact with the flat lining epithelium, thus allowing gases to pass freely by diffusion. — The muscular layers of both chambers are innervated by catecholamine-containing nerve fibres.