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The transcripts of three putative ammonia (NH 3 /NH 4 + ) transporters, Rhesus-like glycoproteins AeRh50-1, AeRh50-2 and Amt/Mep-like AeAmt1 were detected in the anal papillae of larval Aedes aegypti. Quantitative PCR studies revealed 12-fold higher transcript levels of AeAmt1 in anal papillae relative to AeRh50-1, and levels of AeRh50-2 were even lower. Immunoblotting revealed AeAmt1 in anal papillae as a pre-protein with putative monomeric and trimeric forms. AeAmt1 was immunolocalized to the basal side of the anal papillae epithelium where it co-localized with Na +

Teleost fishes are a large and diverse animal group that represent close to 50% of all described vertebrate species. This review consolidates what is known about the claudin (Cldn) family of tight junction (TJ) proteins in teleosts. Cldns are transmembrane proteins of the vertebrate epithelial/endothelial TJ complex that largely determine TJ permeability. Cldns achieve this by expressing barrier or pore forming properties and by exhibiting distinct tissue distribution patterns. So far, ~63 genes encoding for Cldn TJ proteins have been reported in 16 teleost species. Collectively, cldns (or Cldns) are found in a broad array of teleost fish tissues, but select genes exhibit restricted expression patterns. Evidence to date strongly supports the view that Cldns play a vital role in the embryonic development of teleost fishes and in the physiology of tissues and organ systems studied thus far.

SUMMARYThe role of corticosteroid receptors (CRs) in the regulation of gill permeability was examined using a primary cultured trout gill epithelium. The epithelium expressed both glucocorticoid receptors (GR1 and GR2) and a mineralocorticoid receptor (MR), and cortisol treatment significantly increased transepithelial resistance (TER) and decreased paracellular [ 3 H]PEG-4000 flux. Epithelial permeability was unaffected by deoxycorticosterone or aldosterone. The GR antagonist RU486 as well as MR antagonists spironolactone and RU26752 significantly reduced, but did not completely block, the effects of cortisol. The MR antagonist eplerenone was without effect. Only RU486 + spironolactone or RU486 + RU26752 treatment completely suppressed the effects of cortisol. On its own, RU486 had cortisol-like effects which could be blocked by spironolactone, suggesting that although RU486 is a GR antagonist, in this system it may also have agonistic properties that are mediated through the MR. The GR agonist dexamethasone increased TER and reduced [ 3 H]PEG-4000 flux across cultured epithelia and was unaffected by MR antagonists. Therefore, alterations in transcript abundance of select tight junction (TJ) proteins were examined in response to cortisol, dexamethasone (a GR agonist) and RU486 (as a MR agonist). Occludin and claudin-7, -8d, -12 and -31 mRNA were significantly elevated in response to cortisol, dexamethasone or RU486 treatment. Claudin-3a mRNA was significantly elevated in response to cortisol or dexamethasone only, and claudin-28b and -30 mRNA were significantly altered following cortisol or RU486 treatment only. The data support a role for the GRs and MR in regulating gill permeability and suggest that TJ proteins are responsive to cortisol through both or individual CR types.

SUMMARYTight junctions (TJs) are an integral component of models illustrating ion transport mechanisms across fish epithelia; however, little is known about TJ proteins in fishes. Using immunohistochemical methods and Western blot analysis, we examined the localization and expression of occludin, a transmembrane TJ protein, in goldfish tissues. In goldfish gills, discontinuous occludin immunostaining was detected along the edges of secondary gill lamellae and within parts of the interlamellar region that line the lateral walls of the central venous sinus. In the goldfish intestine, occludin immunolocalized in a TJ-specific distribution pattern to apical regions of columnar epithelial cells lining the intestinal lumen. In the goldfish kidney, occludin was differentially expressed in discrete regions of the nephron. Occludin immunostaining was strongest in the distal segment of the nephron, moderate in the collecting duct and absent in the proximal segment. To investigate a potential role for occludin in the maintenance of the hydromineral balance of fishes, we subjected goldfish to 1, 2 and 4 weeks of food deprivation, and then examined the endpoints of hydromineral status, Na + ,K + -ATPase activity and occludin protein expression in the gills, intestine and kidney. Occludin expression altered in response to hydromineral imbalance in a tissue-specific manner suggesting a dynamic role for this TJ protein in the regulation of epithelial permeability in fishes.

Permeability properties of the goldfish gill epithelium were examined in vivo and in vitro following exposure to ion-poor water (IPW) conditions. In gill tissue of IPW-acclimated goldfish, transcript abundance of tight junction (TJ) proteins occludin, claudin-b, -d, -e, -h, -7, and -8d increased, whereas ZO-1 and claudin 12 mRNA decreased and claudin-c was unaltered. In association with these changes, TJ depth increased among gill pavement cells (PVCs) and gill PVCs and mitochondria-rich cells (MRCs). PVC and MRC gill cell fractions were isolated using Percoll. Transcripts encoding for occludin, claudin-b, -c, -d, -e, -h, -7, -8d, -12, and ZO-1 were present in both fractions. After IPW acclimation, occludin, claudin-b and -e, and ZO-1 mRNA abundance increased in both fractions. In contrast, claudin-8d mRNA abundance increased in PVCs only while claudin-h decreased in MRCs. Gill permeability was examined using primary cultured goldfish PVC epithelia supplemented with serum derived from IPW-acclimated goldfish. IPW serum supplementation increased transepithelial resistance, reduced [(3)H]PEG-4000 permeability, and enhanced epithelial integrity during in vitro IPW exposure. IPW serum increased mRNA abundance of occludin, claudin-8d and -e in vitro. Using small interfering RNA, we found that occludin abundance was decreased in cultured gill epithelia, resulting in an increase in [(3)H]PEG-4000 flux. As occludin increased in the gills of IPW-acclimated fish as well as cultured gill epithelia exposed to IPW serum, results suggest that occludin is a barrier-forming TJ protein in fish gill epithelia. These studies support the idea that TJ proteins play an important role in regulating gill permeability in IPW.

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