Christian K. Tipsmark

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.

Nitric oxide synthase in the gill of Atlantic salmon : colocalization with and inhibition of Na+,K+-ATPase

SUMMARY We investigated the relationship between nitric oxide (NO) and Na+,K+-ATPase (NKA) in the gill of anadromous Atlantic salmon. Cells containing NO-producing enzymes were revealed by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, which can be used as an indicator of NOS activity, i.e. NO production. Antibodies against the two constitutive NOS isoforms, neuronal and endothelial NOS, both produced immunoreactivity restricted to large cells at the base and along the secondary lamellae. NADPHd-positive cells showed a corresponding distribution. Antibodies against the inducible NOS isoform only labeled small cells located deep in the filament. Using in situ hybridization and NKA immunoreactivity, cells expressing Na+,K+-ATPaseα -subunit mRNA were found to have a similar distribution to the NOS cells. Double labeling for NOS immunoreactivity and NKA α-subunit mRNA revealed cellular colocalization of NKA α-subunit mRNA and nNOS protein in putative chloride cells at the base of the lamellae and interlamellar space. Along the lamellae, some NOS- or NKA-immunoreactive cells possessed a relatively lower expression of NKA α-subunit mRNA in smolts. A clear increase in NADPHd staining in the gill was demonstrated from parr to smolt. The regulatory role of NO on gill NKA activity was studied in vitro using sodium nitroprusside (SNP; 1 mmol l-1) and PAPA-NONOate (NOC-15; 0.5 mmol l-1) as NO donors. Both SNP and NOC-15 inhibited gill NKA activity by 30% when compared to controls. The study shows that NO systems are abundant in the gill of Atlantic salmon, that NO may be produced preferentially by a constitutive NOS isoform, and suggests that NO influence on gill functions is mediated via intracellular, possibly both auto/paracrine, inhibition of Na+,K+-ATPase activity in chloride cells. Furthermore, the increase in NADPHd in the gill during smoltification suggests a regulatory role of NO in the attenuation of the smoltification-related increase in Na+,K+-ATPase activity prior to entering seawater.

Claudin-15 and -25b expression in the intestinal tract of Atlantic salmon in response to seawater acclimation, smoltification and hormone treatment.

In seawater fishes, osmotic homeostasis requires uptake of ions and water in the intestine and these processes are governed by the combined trans- and paracellular pathways. The current study examined mRNA expression of two tight junction proteins (claudin-15 and -25 b) predominantly expressed in the intestine of Atlantic salmon. We examined the response in pyloric caecae, middle and posterior intestine to seawater challenge, during smoltification and after injection with osmoregulatory hormones. Seawater (SW) transfer elevated levels of claudin-15 and -25 b while no change was induced throughout the smolt stage. In freshwater, cortisol and growth hormone inhibited claudin-15 expression in the two anterior segments. Claudin-25 b was elevated in all intestinal segments by growth hormone, while cortisol had an inhibitory effect. In seawater, prolactin and cortisol inhibited claudin expression. The data suggest that claudin expression is involved in the reorganisation of intestinal epithelium and possibly change paracellular permeability during SW acclimation. The lack of preparatory change during smoltification suggests that this process is not completed during smolt development. The effects of the tested hormones cannot explain the sum of changes induced by salinity, which, like the smoltification data, suggests the importance of additional factors and possibly contact with the imbibed SW per se.

Effects of cortisol, growth hormone and prolactin on gill claudin expression in Atlantic salmon.

We recently showed that a series of tight junction proteins of the claudin family are regulated in the gill of salmon during salinity acclimation. The aim of the present study was to investigate the role of cortisol, growth hormone (GH) and prolactin (PRL) on regulation of expression of these isoforms. Experiments on primary cultures of gill tissue showed that cortisol stimulates claudin 10e, 27a and 30 mRNA levels while no significant effects were observed on claudin 28a and 28b. The associated receptor signalling pathway was examined using glucocorticoid and mineralocorticoid receptor antagonists RU486 and spironolactone, respectively. The observed in vitro responses were blocked by RU486, suggesting the involvement of a glucocorticoid type receptor. Injections of FW salmon with cortisol increased the expression of claudin 10e, 27a, and 30 but did not affect claudin 28a and 28b significantly. While GH had no effect on its own, the combination of GH and cortisol reduced claudin 28b levels. Injection of SW salmon with PRL selectively increased the expression of claudin 28a but had no effect on the other examined isoforms. The data shows that FW- (27a and 30) and SW-induced (10e) claudins are all stimulated by cortisol while the major osmoregulatory hormones GH and PRL had no effect on these salinity sensitive isoforms. This suggests that other hormones and/or osmotic conditions interact with cortisol to determine claudin composition in the gill.

Regulation of Na+/K+-ATPase activity by nitric oxide in the kidney and gill of the brown trout (Salmo trutta).

SUMMARY In teleost fish, successful osmoregulation involves controlled ion transport mechanisms in kidney and gill epithelia. In this study, the effect of nitric oxide (NO) on Na+/K+-ATPase was investigated in vitro in these two tissues in brown trout (Salmo trutta) acclimated to freshwater. The NO donor sodium nitroprusside (SNP) inhibited in situ Na+/K+-ATPase activity, measured as ouabain-sensitive Rb+ uptake, in both samples of kidney and gill tissue and in isolated gill cells. The effect was dose-dependent in both tissues, with a maximal observed inhibition of approximately 40–50% (1 mmol l –1 SNP). The time-course of inhibition revealed a maximum effect with 10 min pre-incubation. The effect of SNP was reproduced with another NO donor, papa-nonoate (NOC-15; 200 μmol l–1), and was prevented by the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 1 mmol l–1). To further investigate the mechanism of the NO effect, whole-tissue Na+ and K+ levels were analysed. In kidney, SNP (1 mmol l–1) led to an increase in tissue Na+ levels and a decrease in K+ levels in a 3:2 ratio. In gill tissue, no change in either ion was observed. These observations indicate that the effect on Na+/K+-ATPase is direct rather than due to a decrease in intracellular Na+, its rate-limiting substrate. SNP elevated the level of cyclic GMP (cGMP) in both kidney and gill tissue. Dibutyryl cyclic GMP (db-cGMP; 1 mmol l–1) also inhibited Na+/K+-ATPase activity in both tissues. Hence, a possible mechanism may involve the cGMP-activated kinase, even though other mechanisms cannot be excluded.

Physiological response in the European flounder (Platichthys flesus) to variable salinity and oxygen conditions

Physiological mechanisms involved in acclimation to variable salinity and oxygen levels and their interaction were studied in European flounder. The fish were acclimated for 2 weeks to freshwater (1‰ salinity), brackish water (11‰) or full strength seawater (35‰) under normoxic conditions (water Po2 = 158 mmHg) and then subjected to 48 h of continued normoxia or hypoxia at a level (Po2 = 54 mmHg) close to but above the critical Po2. Plasma osmolality, [Na+] and [Cl−] increased with increasing salinity, but the rises were limited, reflecting an effective extracellular osmoregulation. Muscle water content was the same at all three salinities, indicating complete cell volume regulation. Gill Na+/K+-ATPase activity did not change with salinity, but hypoxia caused a 25% decrease in branchial Na+/K+-ATPase activity at all three salinities. Furthermore, hypoxia induced a significant decrease in mRNA levels of the Na+/K+-ATPase α1-subunit, signifying a reduced expression of the transporter gene. The reduced ATPase activity did not influence extracellular ionic concentrations. Blood [Hb] was stable with salinity, and it was not increased by hypoxia. Instead, hypoxia decreased the erythrocytic nucleoside triphosphate content, a common mechanism for increasing blood O2 affinity. It is concluded that moderate hypoxia induced an energy saving decrease in branchial Na+/K+-ATPase activity, which did not compromise extracellular osmoregulation.

Developmental Profile of Claudin‐3, ‐5, and ‐16 Proteins in the Epithelium of Chick Intestine

Proteins in the claudin family are a main component of tight junctions and form a seal that modulates paracellular transport in intestinal epithelium. This research tests the hypothesis that claudins 3, 5, and 16 will appear in the epithelium of embryonic intestine during functional differentiation. Immunohistochemistry is utilized to explore the developmental patterns of claudin‐3, ‐5, and ‐16 proteins in the epithelium of embryonic chick intestine from 9 days prior to hatching through the early post‐hatch period. These claudin proteins either changed their cellular localization or first appeared around the time of hatching. After hatching, claudin‐3 expression was prominent in basal–lateral regions of the epithelium along the entire villus, but was absent from crypts. Claudin‐5 was expressed most strongly in the crypt and lower villus epithelium within junctional complexes, whereas immunostaining of claudin‐16 was localized within goblet cells of the upper villus region. The relative mRNA levels of claudin‐3, ‐5, and ‐16 showed similar patterns; transcript levels rose between 18 and 20 days of development, then dropped by 2 days post‐hatch. Results of this work indicate that the claudin proteins assume their final locations within the epithelium around the time of hatching, suggesting that in addition to their known barrier and fence functions within tight junctions, these claudins may have additional roles in the differentiation and/or physiological function of chick intestine. The localization of claudin‐16 to goblet cells and its distribution in the more mature cells of the upper villus region suggest an unexpected role in goblet cell maturation and mucus secretion. Anat Rec 293:1175–1183, 2010.