Drinking and Water Handling in the Medaka Intestine: A Possible Role of Claudin-15 in Paracellular Absorption?

When euryhaline fish move between fresh water (FW) and seawater (SW), the intestine undergoes functional changes to handle imbibed SW. In Japanese medaka, the potential transcellular aquaporin-mediated conduits for water are paradoxically downregulated during SW acclimation, suggesting paracellular transport to be of principal importance in hyperosmotic conditions. In mammals, intestinal claudin-15 (CLDN15) forms paracellular channels for small cations and water, which may participate in water transport. Since two cldn15 paralogs, cldn15a and cldn15b, have previously been identified in medaka, we examined the salinity effects on their mRNA expression and immunolocalization in the intestine. In addition, we analyzed the drinking rate and intestinal water handling by adding non-absorbable radiotracers, 51-Cr-EDTA or 99-Tc-DTPA, to the water. The drinking rate was >2-fold higher in SW than FW-acclimated fish, and radiotracer experiments showed anterior accumulation in FW and posterior buildup in SW intestines. Salinity had no effect on expression of cldn15a, while cldn15b was approximately 100-fold higher in FW than SW. Despite differences in transcript dynamics, Cldn15a and Cldn15b proteins were both similarly localized in the apical tight junctions of enterocytes, co-localizing with occludin and with no apparent difference in localization and abundance between FW and SW. The stability of the Cldn15 protein suggests a physiological role in water transport in the medaka intestine.

Prolactin controls Na+,Cl- cotransporter via Stat5 pathway in the teleost gill.

In some freshwater fish species, the control of gill Na, Cl cotransporter (Ncc2b) by prolactin appears to be instrumental to ionic homeostasis. This study was carried out to examine the signaling pathways involved in prolactin-mediated salt retention using gill explants from Japanese medaka (Oryzias latipes). Ovine prolactin induced a concentration-dependent stimulation of ncc2b with significant effects of 10, 100 and 1000 ng of hormone per mL media (2-6 fold). To understand the molecular mechanisms mediating prolactin control of gill function, we analyzed effects on signaling pathways known to be involved in the hormones action in other systems, namely Stat5, Akt and Erk1/2. Their activation was examined in a time course and concentration response experiment. Prolactin (1 µg mL-1) induced a rapid phosphorylation (stimulation) of Stat5 (10 min) that reached a plateau after 30 min and was maintained for at least 120 min. The effect of prolactin on Stat5 phosphorylation was concentration-dependent (4-12 fold). No activation of Akt or Erk1/2 was observed in either experiment. The Stat5 activation was further investigated in localization studies that demonstrated strong nuclear expression of phosphorylated Stat5 in prolactin-treated gill ionocytes. Using specific inhibitors, we analyzed the signalling pathways mediating prolactin induction of gill ncc2b. Co-incubation experiments showed that Stat5 inhibition blocked prolactin's stimulation of ncc2b expression, while PI3K-Akt and Mek1/2-Erk1/2 pathway inhibitors had no effect. These findings show that ncc2b expression is dependent on prolactin's downstream activation of Stat5 and its subsequent nuclear translocation within branchial ionocytes.

Prolactin and cortisol regulate branchial claudin expression in Japanese medaka.

Several gill claudin (Cldn) tight junction proteins in Japanese medaka are regulated by salinity (cldn10 paralogs and cldn28b), while others are constitutively expressed (cldn27a, cldn28a and cldn30c). The role of the endocrine system in this regulation has yet to be understood. The in vitro effects of cortisol and prolactin on cldn expression in gill explant cultures were investigated in medaka. ncc2b and cftr were used as markers of specific ionocytes associated with freshwater- and seawater-acclimation, respectively. Concentration-response experiments were performed by overnight incubation with 0, 0.1, 1 and 10µgmL-1 cortisol or 0, 0.01, 0.1 and 1µgmL-1 ovine prolactin. Cortisol significantly up-regulated cftr, ncc2b, cldn10 paralogs, cldn27a and cldn30c from 1.2- to 5-fold control levels at 10µgmL-1. Cortisol had no effect on cldn28a and cldn28b. Prolactin had a concentration-dependent effect, decreasing expression of cftr (1µgmL-1, 2.2-fold) while increasing ncc2b (from 0.1µgmL-1, 6-7-fold). Prolactin up-regulated expression of 3 cldns: cldn28b (0.1 and 1µgmL-1), cldn10c and cldn10f (1µgmL-1), with up to 2-, 2.5- and 2-fold of control level, respectively. A combination experiment with both hormones showed that they act in synergy on cldn28b and have an additive effect on cftr, ncc2b, cldn10c and cldn10f. Our results showed that cortisol and prolactin are essential to maintain the expression of specific branchial claudins. This work also provides evidence that both hormones act directly on gill of medaka to modulate determinants of paracellular ion movement.

Does Japanese medaka (Oryzias latipes) exhibit a gill Na(+)/K(+)-ATPase isoform switch during salinity change?

Some euryhaline teleosts exhibit a switch in gill Na(+)/K(+)-ATPase (Nka) α isoform when moving between fresh water (FW) and seawater (SW). The present study tested the hypothesis that a similar mechanism is present in Japanese medaka and whether salinity affects ouabain, Mg(2+), Na(+) and K(+) affinity of the gill enzyme. Phylogenetic analysis classified six separate medaka Nka α isoforms (α1a, α1b, α1c, α2, α3a and α3b). Medaka acclimated long-term (>30 days) to either FW or SW had similar gill expression of α1c, α2, α3a and α3b, while both α1a and α1b were elevated in SW. Since a potential isoform shift may rely on early changes in transcript abundance, we conducted two short-term (1-3 days) salinity transfer experiments. FW to SW acclimation induced an elevation of α1b and α1a after 1 and 3 days. SW to FW acclimation reduced α1b after 3 days with no other α isoforms affected. To verify that the responses were typical, additional transport proteins were examined. Gill ncc and nhe3 expression were elevated in FW, while cftr and nkcc1a were up-regulated in SW. This is in accordance with putative roles in ion-uptake and secretion. SW-acclimated medaka had higher gill Nka V max and lower apparent K m for Na(+) compared to FW fish, while apparent affinities for K(+), Mg(2+) and ouabain were unchanged. The present study showed that the Japanese medaka does not exhibit a salinity-induced α isoform switch and therefore suggests that Na(+) affinity changes involve altered posttranslational modification or intermolecular interactions.

Functional dynamics of claudin expression in Japanese medaka (Oryzias latipes): Response to environmental salinity.

Salinity regulation of 13 claudin paralogs was investigated in osmoregulatory organs of euryhaline Japanese medaka. They were identified by blast-search in the medaka genome database based on representation in osmoregulatory organs of other teleosts. Our hypothesis was that, because of their sequence similarities to mammalian orthologs previously characterized as barrier- and ion-selective channel-forming proteins, these paralogs would respond to salinity according to expected modulation of osmoregulatory function. Cldn10c, -10d, -10e, -10f, -27a, -28a, -28b and -30c had 4- to 100-fold higher expression in gill than other examined organs. Two splice variants of cldn10b were predominantly expressed in kidney, while cldn15a, -15b and -25 were found mainly in intestine. In gills, cldn27a, -28a, -28b and -30c did not change between fresh water (FW) and seawater (SW)-acclimated fish, while cldn10c, -10d, -10e, and -10f were most abundant in SW. Short-term SW transfer induced up-regulation of cldn10 gill paralogs after 1 day, decrease in cldn28b and no difference for cldn27a, -28a and -30c. The reverse pattern was observed after FW transfer of SW medaka. Intestinal cldn15a and -25 did not differ between FW and SW fish. However, cldn15b was 10-fold higher in FW than SW, suggesting a role in functional modulation of the intestine related to water and salt transport. In kidney, cldn10bs were elevated in SW fish, suggesting a role in paracellular ion transport in the marine nephron. Based on in silico analysis, most gill Cldn10s were predicted to form cation pores, whereas Cldn27a, 28a, 28b and 30c may increase epithelial resistance.

Transepithelial resistance and claudin expression in trout RTgill-W1 cell line: effects of osmoregulatory hormones.

In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1-2 days (20-80Ω⋅cm(2)), which was then maintained for more than 6 days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6h. Treatment with ethylenediaminetetraacetic acid induced a decrease in TER to <15% of pre-treatment level. Cortisol elevated TER after 12-72 h in a concentration-dependent manner, and this increase was antagonized by growth hormone (Gh). The effects of three osmoregulatory hormones, Gh, prolactin, and cortisol, on the mRNA expression of three tight junction proteins were examined: claudin-10e (Cldn-10e), Cldn-30, and zonula occludens-1 (Zo-1). The expression of cldn-10e was stimulated by all three hormones but with the strongest effect of Gh (50-fold). cldn-30 expression was stimulated especially by cortisol (20-fold) and also by Gh (4-fold). Finally, zo-1 was unresponsive to hormone treatment. Western blot analysis detected Cldn-10e and Cldn-30 immunoreactive proteins of expected molecular weight in samples from rainbow trout gills but not from RTgill-W1 cultures, possibly due to low expression levels. Collectively, these results show that the RTgill-W1 cell layers have tight junctions between cells, are sensitive to hormone treatments, and may provide a useful model for in vitro study of some in vivo gill phenomena.

Behavioural and transcriptional changes in the amphipod Echinogammarus marinus exposed to two antidepressants, fluoxetine and sertraline.

In the past decade, there have been increasing concerns over the effects of pharmaceutical compounds in the aquatic environment, however very little is known about the effects of antidepressants such as the selective serotonin re-uptake inhibitors (SSRIs). Many biological functions within invertebrates are under the control of serotonin, such as reproduction, metabolism, moulting and behaviour. The effects of serotonin and fluoxetine have recently been shown to alter the behaviour of the marine amphipod, Echinogammarus marinus (Leach, 1815). The purpose of this study was to observe behavioural and transcriptional modifications in this crustacean exposed to the two most prescribed SSRIs (fluoxetine and sertraline) and to develop biomarkers of neurological endocrine disruption. The animals were exposed to both drugs at environmentally relevant concentrations from 0.001 to 1µg/L during short-term (1h and 1day) and medium-term (8 days) experiments. The movement of the amphipods was tracked using the behavioural analysis software during 12min alternating dark/light conditions. The behavioural analysis revealed a significant effect on velocity which was observed after 1h exposure to sertraline at 0.01µg/L and after 1 day exposure to fluoxetine as low as 0.001µg/L. The most predominant effect of drugs on velocity was recorded after 1 day exposure for the 0.1 and 0.01µg/L concentrations of fluoxetine and sertraline, respectively. Subsequently, the expression (in this article gene expression is taken to represent only transcription, although it is acknowledged that gene expression can also be regulated at translation, mRNA and protein stability levels) of several E. marinus neurological genes, potentially involved in the serotonin metabolic pathway or behaviour regulation, were analysed in animals exposed to various SSRIs concentrations using RT-qPCR. The expression of a tryptophan hydroxylase (Ph), a neurocan core protein (Neuc), a Rhodopsin (Rhod1) and an Arrestin (Arr) were measured following exposure to fluoxetine or sertraline for 8 days. The levels of Neuc, Rhod1 and Arr were significantly down-regulated to approximately 0.5-, 0.29- and 0.46-fold, respectively, for the lower concentrations of fluoxetine suggesting potential changes in the phototransduction pathway. The expression of Rhod1 tended to be up-regulated for the lower concentration of sertraline but not significantly. In summary, fluoxetine and sertraline have a significant impact on the behaviour and neurophysiology of this amphipod at environmentally relevant concentrations with effects observed after relatively short periods of time.