In vertebrates, tight junction (TJ) proteins play an important role in epithelium formation and development, the maintenance of tissue integrity and regulation of TJ permeability. In this study, primary cultured model gill epithelia composed of pavement cells (PVCs) were used to examine TJ protein transcript abundance during the development of epithelium confluence and epithelium resistive properties. Differences in TJ protein expression patterns and transcript abundance between gill models composed of PVCs and models composed of PVCs and mitochondrion-rich cells (MRCs) were also examined. Marked alterations in TJ protein transcript abundance were observed as cells developed to confluence in flask-cultured model gill epithelia. In contrast, during the formation of tissue resistance in insert-cultured epithelia (i.e. epithelia cultured on a permeable substrate), changes in TJ protein mRNA abundance were conservative, despite paracellular marker flux decreasing by orders of magnitude. In both cases significant changes in claudin-8b, -8d, -27b, -28b and - 32a transcript abundance were observed, suggesting that temporal alterations in the abundance of these genes are important end points of model gill epithelium integrity. When MRCs were present in cultured gill models, the mRNA abundance of several TJ proteins significantly altered and claudin-10c, -10d and - 33b were only detected in preparations that included MRCs. These data provide insight into the role of select TJ proteins in the formation and development of gill epithelia and the maintenance of gill barrier properties. In addition, observations reveal a heterogeneous distribution of claudin TJ proteins in the gill epithelial cells of rainbow trout.

SUMMARY Gill Na + /K + -ATPase (NKA) in teleost fishes is involved in ion regulation in both freshwater and seawater. We have developed and validated rabbit polyclonal antibodies specific to the NKA α1a and α1b protein isoforms of Atlantic salmon ( Salmo salar Linnaeus), and used western blots and immunohistochemistry to characterize their size, abundance and localization. The relative molecular mass of NKA α1a is slightly less than that for NKA β1b. The abundance of gill NKA α1a was high in freshwater and became nearly undetectable after seawater acclimation. NKA α1b was present in small amounts in freshwater and increased 13-fold after seawater acclimation. Both NKA isoforms were detected only in chloride cells. NKA α1a was located in both filamental and lamellar chloride cells in freshwater, whereas in seawater it was present only as a faint background in filamental chloride cells. In freshwater, NKA α1b was found in a small number of filamental chloride cells, and after seawater acclimation it was found in all chloride cells on the filament and lamellae. Double simultaneous immunofluorescence indicated that NKA α1a and α1b are located in different chloride cells in freshwater. In many chloride cells in seawater, NKA α1b was present in greater amounts in the subapical region than elsewhere in the cell. The combined patterns in abundance and immunolocalization of these two isoforms can explain the salinity-related changes in total NKA and chloride cell abundance. The results indicate that there is a freshwater and a seawater isoform of NKA α-subunit in the gills of Atlantic salmon and that they are present in distinct chloride cells.

SUMMARY In the kidneys of mammals, cyclooxygenase type 2 (COX2) is expressed in medullary interstitial cells, the macula densa and epithelial cells of the cortical thick ascending limb where it generates prostaglandins that regulate hormone secretion, inhibit ion transport, and support cell survival during salt loading and dehydration. In teleosts, the gills are in direct contact with an aquatic environment and are the dominant site of osmoregulation. During transfers between salinities, specialized cells in the gills (chloride cells) rapidly regulate NaCl secretion for systemic osmoregulation while they simultaneously are exposed to acute osmotic shock. This study was conducted to determine if COX2 is expressed in the gills, and if so, to evaluate its function in cellular and systemic osmoregulation. Degenerate primers, reverse transcription–PCR and rapid amplification of cDNA ends were used to deduce the complete cDNA sequence of a putative COX2 enzyme from the gills of the euryhaline killifish ( Fundulus heteroclitus ). The 2738 base pair cDNA includes a coding region for a 610 amino acid protein that is over 70%identical to mammalian COX2. A purified antibody generated against a conserved region of mouse COX2 labeled chloride cells, suggesting that the enzyme may control NaCl secretion as an autocrine agent. Real-time PCR was then used to demonstrate that mRNA expression of the COX2 homologue was threefold greater in gills from chronic seawater killifish than in gills from chronic freshwater killifish. Expression of Na + /K + /2Cl – cotransporter and the cystic fibrosis transmembrane conductance regulator were also greater in seawater, suggesting that chronic COX2 expression in the gills is regulated in parallel to the key ion transporters that mediate NaCl secretion. Real-time PCR was also used to demonstrate that acute transfer from seawater to freshwater and from freshwater to seawater led to rapid, transient inductions of COX2 expression. Together with previous physiological evidence,the present molecular and immunological data suggest that constitutive branchial COX2 expression is enhanced in seawater, where prostaglandins can regulate NaCl secretion in chloride cells. Our data also suggest that branchial COX2 expression may play a role in cell survival during acute osmotic shock.

SUMMARY We have recently established a unique in vitro experimental model for mitochondrion-rich cell (MRC) research, a `yolk-ball' incubation system,in which the yolk sac is separated from the embryonic body of Mozambique tilapia embryos and subjected to in vitro incubation. To evaluate the ion-transporting property of the yolk balls, we examined Cl – content and turnover in yolk balls incubated in freshwater and seawater for 48 h, and distribution patterns of three ion transporters,Na + /K + -ATPase,Na + /K + /2Cl – cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR), in MRCs in the yolk-sac membrane. The Cl – turnover rate measured by whole-body influx of 36 Cl – was about 60 times higher in yolk balls in seawater than in freshwater, while there was no essential difference in Cl – content between them. Na + /K + -ATPase-immunoreactive MRCs were larger in yolk balls from seawater than yolk balls from freshwater. Distribution patterns of ion-transporting proteins allowed us to classify MRCs in freshwater yolk balls into three types: cells showing only basolateral Na + /K + -ATPase, cells showing basolateral Na + /K + -ATPase and apical NKCC, and cells showing basolateral Na + /K + -ATPase and basolateral NKCC. The seawater yolk balls, on the other hand, were characterized by the appearance of MRCs possessing basolateral Na + /K + -ATPase,basolateral NKCC and apical CFTR. Those seawater-type MRCs were considered to secrete Cl – through the CFTR-positive apical opening to cope with diffusional Cl – influx. These findings indicate that the yolk balls preserve the Cl – transporting property of intact embryos, ensuring the propriety of the yolk ball as an in vitro experimental model for the yolk-sac membrane that contains MRCs.

SUMMARY Mozambique tilapia Oreochromis mossambicus embryos were transferred from freshwater to seawater and vice versa , and short-term changes in the localization of three major ion transport proteins,Na + /K + -ATPase,Na + /K + /2Cl - cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) were examined within mitochondrion-rich cells (MRCs) in the embryonic yolk-sac membrane. Triple-color immunofluorescence staining allowed us to classify MRCs into four types: type I, showing only basolateral Na + /K + -ATPase staining; type II, basolateral Na + /K + -ATPase and apical NKCC; type III, basolateral Na + /K + -ATPase and basolateral NKCC; type IV, basolateral Na + /K + -ATPase,basolateral NKCC and apical CFTR. In freshwater, type-I, type-II and type-III cells were observed. Following transfer from freshwater to seawater, type-IV cells appeared at 12 h and showed a remarkable increase in number between 24 h and 48 h, whereas type-III cells disappeared. When transferred from seawater back to freshwater, type-IV cells decreased and disappeared at 48 h, type-III cells increased, and type-II cells, which were not found in seawater, appeared at 12 h and increased in number thereafter. Type-I cells existed consistently irrespective of salinity changes. These results suggest that type I is an immature MRC, type II is a freshwater-type ion absorptive cell, type III is a dormant type-IV cell and/or an ion absorptive cell (with a different mechanism from type II), and type IV is a seawater-type ion secretory cell. The intracellular localization of the three ion transport proteins in type-IV cells is completely consistent with a widely accepted model for ion secretion by MRCs. A new model for ion absorption is proposed based on type-II cells possessing apical NKCC.