The structure of the caudal wall of the zebrafish (Danio rerio) swim bladder: evidence of localized lamellar body secretion and a proximate neural plexus.

In this study, we present a morphological description of the fine structure of the tissues composing the caudal tip of the adult zebrafish swim bladder and an immunochemical survey of the innervation at this site. The internal aspect of the caudal tip is lined by an epithelium specialized to secrete surfactant into the lumen as evinced by the exocytosis of lamellar bodies. The sole innervation to this region consists of a neural plexus, present on the external surface, of nitric oxide synthase-positive (nNOS) neuronal cell bodies that are contacted by axon terminals, some containing neuropeptide Y and vasoactive intestinal polypeptide. As the specialized epithelium and neural plexus are coincident and of common extent, we suggest that the morphological relationship between the two elements allows the nervous system to affect surfactant processing, possibly through a paracrine mechanism.

Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish.

The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR(2)) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on ß-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC(50) values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos(3) failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells.

[Influence of electroacupuncture on human recombination tumor necrosis factor-alpha induced cycloxygenase expression in air sac model rats].

OBJECTIVE: To explore the intervention of electro-acupuncture (EA) on cycloxygenase (COX) mRNA and protein expression induced by human recombination tumor necrosis factor-alpha (hrTNF). METHODS: Air sac model was established by implanted an autoclaved teflon chamber into back of rat (SPF grade). Ten days after modeling, 90 qualified rats, in them inflammation not detected, were randomly divided into three groups, the control group, the TNF model (model) group and the TNF +EA (TE) group, with 30 cases in each. To rats in the control group, 1 mL of saline was injected into the sac, but to those in the model and TE groups, 2 ng/mL hrTNF in a volume of 1 mL was used instead to induce local inflammatory responses. Immediately after then, EA (2 Hz, 5 mA and persistent waves) was applied to bilateral Quchi points (LI11) of rats in the TE group for 30 min. Fluid in the sac was drawn out at various time points (1, 5 and 24 h) after injection to measure the COX-2 mRNA expression by RT-PCR and COX-2 protein expression by Western blot. RESULTS: Level of COX-2 mRNA expression in the control group was significantly different to that in the other two groups (P < 0.05 or P < 0.01). COX-2 mRNA expression at the 24th hour and COX-2 protein expression at the 1st hour were significantly lower in the TE group than those in the model group (P < 0.05 and P < 0.01) respectively. CONCLUSION: EA could effectively intervene the mRNA and protein expression of COX-2 induced by hrTNF, which is possibly by way of pro-inflammatory cytokine regulating.

Localization of the vacuolar-type ATPase in swimbladder gas gland cells of the European eel (Anguilla anguilla).

The vacuolar ATPase is a multifunctional enzyme that consists of several subunits. Subunit B is part of the catalytic domain of the enzyme and is present in two isoforms in fish as well as in mammals. Possibly, these two isoforms - vatB1 (kidney isoform) and vatB2 (brain isoform) - serve different functions. A localization of the two isoforms was attempted in swimbladder gas gland cells of the European eel Anguilla anguilla by immunohistochemistry. Two antibodies were produced by immunization of rabbits with synthetic peptides. Specificity of the antibodies, on the one hand, an isoform-specific antibody for vatB1 and, on the other hand, an antibody that recognizes both isoforms (vatB1 and vatB2), was confirmed by western blot analysis using recombinant proteins produced in a bacterial expression system. The immunohistochemical localization with the antibody directed against both isoforms of the B subunit revealed a positive staining in apical membranes of swimbladder gas gland cells as well as in the basolateral membranes. Significant staining was observed in vesicles located near the apical membrane. Staining with the vatB1-specific antibody resulted in a similar picture in the apical region of the cells. In contrast to the staining with the first antibody, only a poor signal was observed in the basal region. The nature of the vesicles in the apical region of the gas gland cells was determined by using an antibody directed against surfactant protein D.

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.

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.

Catalase, glutathione peroxidase, and superoxide dismutase in the rete mirabile and gas gland epithelium of six species of marine fishes.

Rete mirabile and gas gland epithelium from the swim bladders of six species of marine fishes were assayed for catalase, glutathione peroxidase, and superoxide dismutase activity. Correlation of the results of these assays with measurements of the concentration of oxygen in the lumen of the normal steady state swim bladders revealed that swim bladders in species containing higher levels of oxygen also exhibited higher levels of superoxide dismutase activity in the rete mirabile/gas gland epithelium region. There appeared to be no correlation between oxygen concentration and the level of catalase or glutathione peroxidase activity. Induction of the inflatory reflex in Opsanus tau by a single deflation of the swim bladder resulted in an increase in the percent of oxygen in the swim bladder lumen 18 to 24 hours later, but this was not accompanied by any significant increases in antioxidant enzyme activity. Swim bladders that were deflated three times at 24-hour intervals showed further increases in oxygen concentration at the end of the 72-hour period but no alteration in superoxide dismutase activity.