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 -25b) 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 -25b 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-25b 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.     

Differential expression of absorptive cation-chloride-cotransporters in the intestinal and renal tissues of the European eel (Anguilla anguilla)

Duplicate pairs of isoforms of each of the NKCC2 and the NCC absorptive cation-chloride-cotransporters have been isolated from the European eel. As with mammalian NKCC2, NKCC2alpha isoform mRNA expression was restricted to renal tissues, whereas NKCC2beta isoform expression was present in intestine and urinary bladder. Similar to mammalian NCC, NCCalpha mRNA expression was also found in the kidney, whereas, expression of NCCbeta mRNA was found at low levels in a number of tissues but particularly in intestine. Following 3 weeks of transfer of yellow or silver (adult life stages) eels from freshwater (FW) to seawater (SW), renal mRNA expression of NKCC2alpha did not change whereas NCCalpha expression was reduced although only significantly in silver eels. This suggests that any changes in renal sodium chloride re-absorption in SW-acclimated fish may be due to decreased NCCalpha cotransporter activity rather than the result of suppression of NKCCalpha cotransporter activity. Intestinal mRNA expression of NKCC2beta generally increased following SW acclimation, although maximal increases occurred later in yellow (7 days) than silver (2 days) eels. Average levels of NKCC2beta mRNA abundance in the middle intestine were 89% of those in the anterior, and this was reduced to 44% (of the level in the anterior intestine) in posterior intestine/rectum. Expression of NCCbeta was only found in the posterior intestine/rectum. Together these results suggest intestinal sodium chloride absorption may switch from occurring via NKCCbeta to NCCbeta as imbibed fluid travels down the intestine and the concentration of luminal potassium decreases.     

Physiological and molecular endocrine changes in maturing wild sockeye salmon, <Emphasis Type="Italic">Oncorhynchus nerka</Emphasis>, during ocean and river migration

Maturing adult sockeye salmon Oncorhynchus nerka were intercepted while migrating in the ocean and upstream in freshwater over a combined distance of more than 1,300 km to determine physiological and endocrine changes associated with ionoregulation. Sockeye migrating through seawater and freshwater showed consistent declines in gill Na⁺/K⁺-ATPase (NKA) activity, plasma osmolality and plasma chloride concentration. In contrast, plasma sodium concentration became elevated in seawater as fish approached the river mouth and was then restored after sockeye entered the river. Accompanying the movement from seawater to freshwater was a significant increase in mRNA for the NKA α1a subunit in the gill, with little change in the α1b subunit. Potential endocrine signals stimulating the physiological changes during migration were assessed by measuring plasma cortisol and prolactin (Prl) concentrations and quantifying mRNA extracted from the gill for glucocorticoid receptors 1 and 2 (GR1 and GR2), mineralocorticoid receptor (MR), growth hormone 1 receptor (GH1R), and prolactin receptor (PrlR). Plasma cortisol and prolactin concentrations were high in seawater suggesting a preparatory endocrine signal before freshwater entry. Generally, the mRNA expression for GR1, GR2 and MR declined during migration, most notably after fish entered freshwater. In contrast, PrlR mRNA increased throughout migration, particularly as sockeye approached the spawning grounds. A highly significant association existed between gill PrlR mRNA and gill NKA α1a mRNA. GH1R mRNA also increased significantly, but only after sockeye had migrated beyond tidal influence in the river and then again just before the fish reached the spawning grounds. These findings suggest that cortisol and prolactin stimulate ionoregulation in the gill as sockeye salmon adapt to freshwater.     

Water and NaCl absorption by the intestine of the tilapiaSarotherodon mossambicus adapted to fresh water or seawater and the possible role of prolactin and cortisol

Summary Rates of intestinal water, sodium and chloride absorption in tilapia, adapted to fresh water (FW) and seawater (SW), were measured in vitro, using noneverted sacs made from the anterior, middle and posterior intestinal regions. The anterior intestine from SW fish showed considerably less water, sodium and chloride absorption compared with that seen in FW fish. The middle intestine showed either minimal absorption or some secretion in both FW and SW. In the posterior intestine, water absorption was only limitedly affected by SW-adaptation, but sodium and chloride absorption rates were significantly lower in SW fish. Reductions in water absorption were already evident in the anterior intestine 24 h after transfer to 1/3 SW but reached lower levels 3 to 5 days following transfer to 100% SW. Thus, the anterior intestine of tilapia responds to increased environmental salinity by decreasing uptake of ions, whereas the posterior intestine maintains similar water absorption in both FW and SW, although ion absorption is lower in SW.Prolactin administration to SW fish augmented sodium and water absorption in the anterior intestine but had no effect on chloride absorption. In contrast, cortisol administration to FW fish decreased absorption of sodium, chloride and water to levels usually seen in SW fish. The observed effects of these hormones in tilapia intestinal absorption may be confined to the specialized anterior intestinal region in this species; hormonal effects on the rest of the intestine were not examined.     

Regulation of expression of two aquaporin homologs in the intestine of the European eel: effects of seawater acclimation and cortisol treatment

Complementary DNAs encoding homologs of the mammalian aquaglyceroporins (termed AQPe) and aquaporin-1 isoforms (termed AQP1) were isolated from the European eel. The AQP amino acid sequences share 35-54% identity with other known human AQPs. Although AQPe mRNA expression was approximately equivalent along the entire length of the gut, AQP1 expression was the highest in the posterior/rectal segment. Seawater (SW) acclimation increased AQP1 mRNA abundance by 5- and 17-fold in the anterior, 14- and 23-fold in the mid-, and 9- and 7-fold in the posterior/rectal gut regions of yellow and silver eels, respectively. SW acclimation had an effect on AQPe mRNA expression only in the midintestine of silver eels, where a small but significant 1.7-fold increase in abundance was measured. Western blots using an eel AQP1-specific antibody identified the presence of a major immunoreactive 28-kDa protein, primarily within the posterior/rectal segment. A 3-wk SW transfer induced an increase in AQP1 protein abundance in all intestinal segments, with the posterior/rectal region still expressing protein levels approximately 40- and 8-fold higher than the anterior and midsegments, respectively. Strong AQP1 immunofluorescence was detected within the vascular endothelium in both freshwater (FW)- and SW-acclimated eels and in the epithelial apical brush border in the posterior/rectal gut regions of SW-acclimated eels. Cortisol infusion into FW eels had no effect on intestinal AQPe mRNA expression but induced increases in AQP1 mRNA and protein levels. These results provide evidence for the presence of a SW-induced and steroid-regulated AQP water channel pathway within the intestine of the European eel.     

Increased maternal cortisol in late-gestation ewes decreases fetal cardiac expression of 11beta-HSD2 mRNA and the ratio of AT1 to AT2 receptor mRNA

Moderately elevated maternal cortisol levels late in gestation cause enlargement of the fetal sheep heart. We have used quantitative real-time PCR to examine expression of candidate genes in fetal hearts from mothers in whom cortisol levels were increased (by infusion of 1 mg cortisol.kg(-1).day(-1)) or decreased (by adrenalectomy and replacement to 0.5 mg cortisol.kg(-1).day(-1)) from 115 to 130 days gestation. Control ewes were not treated with steroid. Expression of mineralocorticoid receptor (MR), glucocorticoid receptor (GR), 11beta-hydroxysteroid dehydrogenases 1 and 2 (11beta-HSD1 and -2), IGF I and II, IGF receptors 1 and 2 (IGF-1R and IGF-2R), endothelial nitric oxide synthase, VEGF, myotrophin, angiotensinogen, the angiotensin receptors 1 and 2 (AT1R and AT2R), and the angiotensin converting enzymes 1 and 2 were measured. MR mRNA abundance in fetal hearts was found to be similar to that in adult kidney and hippocampus. Although there were no significant changes in most genes, 11beta-HSD2 and IGF-1R expression were significantly decreased in the high cortisol group and 11beta-HSD2 expression negatively correlated to left ventricular wall thickness. There was also a significant change in the ratio of AT receptor expression, with increased AT2R and decreased AT1R in the high cortisol group. MR, GR, and 11beta-HSD1 immunoreactivity was found in cardiomyocytes and cardiac blood vessels in 126-128 day fetal sheep; in contrast 11beta-HSD2 staining was predominantly in blood vessels. These results indicate that cortisol could indeed act in the fetal heart to induce enlargement and suggest that the renin-angiotensin system may play a role.     

Identification and expression of natriuretic peptide receptor type-A and -B mRNA in freshwater and seawater rainbow trout

Natriuretic peptide receptors mediate the physiological response of natriuretic peptide hormones. One of the natriuretic peptide receptor types is the particulate guanylyl cyclase receptors, of which there are two identified: NPR-A and NPR-B. In fishes, these have been sequenced and characterized in eels, medaka, and dogfish shark (NPR-B only). The euryhaline rainbow trout provides an opportunity to further pursue examination of the system in teleosts. In this study, partial rainbow trout NPR-A-like and NPR-B-like mRNA sequences were identified via PCR and cloning. The sequence information was used in real-time PCR to examine mRNA expression in a variety of tissues of freshwater rainbow trout and rainbow trout acclimated to 35 parts per thousand seawater for a period of 10 days. In the excretory kidney and posterior intestine, real-time PCR analysis showed greater expression of NPR-B in freshwater fish than in those adapted to seawater; otherwise, there was no difference in the expression of the individual receptors in fresh water or seawater. In general, the expression of the NPR-A and NPR-B type receptors was quite low. These findings indicate that NPR-A and NPR-B mRNA expression is minimally altered under the experimental regime used in this study.     

Glucocorticoid-mediated attenuation of the hsp70 response in trout hepatocytes involves the proteasome

The physiological implication of elevated cortisol levels on cellular heat-shock protein 70 (hsp70) response was examined using primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. Trout hepatocytes treated with cortisol, the predominant glucocorticoid in teleosts, responded to the heat shock (+15 degrees C for 1 h) with a significant drop in hsp70 accumulation over a 24-h recovery period. [(35)S]methionine incorporation and pulse-chase studies confirmed that this cortisol impact was due to decreased hsp70 synthesis and not enhanced protein breakdown. Cortisol also significantly decreased glucocorticoid receptor (GR) expression in trout hepatocytes. This receptor downregulation was inhibited by the proteasomal inhibitors, lactacystin and MG-132, implying a role for the proteasome in GR downregulation by cortisol. Inhibiting the proteasome did not significantly modify heat-induced hsp70 accumulation in the absence of cortisol but significantly elevated hsp70 expression in the presence of cortisol in heat-shocked trout hepatocytes. Taken together, our results suggest proteasome-mediated GR degradation as a mechanism for the attenuation of hsp70 response by cortisol in heat-shocked hepatocytes.     

Tissue culture of sockeye salmon intestine: functional response of Na+-K+-ATPase to cortisol

A method to culture tissue explants of the intestine from freshwater-adapted sockeye salmon (Oncorhynchus nerka) was developed to assess possible direct effects of cortisol on Na(+)-K(+)-ATPase activity. As judged by several criteria, explants from pyloric ceca and the posterior region of the intestine remained viable during short-term (6-day) culture, although Na(+)-K(+)-ATPase activity declined and basolateral components of the enterocytes were observed to be partially degraded. Addition of cortisol to the culture medium maintained Na(+)-K(+)-ATPase activity (over 2-12 days) above that of control explants and, in some cases, was similar to levels before culture. The response to cortisol was dose dependent (0.001-10 microg/ml). Within the physiological range, the response was specific for cortisol and showed the following hierarchy: dexamethasone >/= cortisol > 11-deoxycortisol > cortisone. Insulin maintained Na(+)-K(+)-ATPase activity over controls in explants of ceca but not posterior intestine. To compare in vivo and in vitro responses, slow-release implants of cortisol (50 microg/g) were administered to salmon for 7 days. This treatment elevated plasma cortisol levels and stimulated Na(+)-K(+)-ATPase activity in both intestinal regions. The results demonstrate that the teleost intestine is a direct target of cortisol, this corticosteroid protects in vitro functionality of Na(+)-K(+)-ATPase, and explants retain cortisol responsiveness during short-term culture.     

Two isoforms of the Na+/K+/2Cl- cotransporter are expressed in the European eel (Anguilla anguilla)

Two cDNA isoforms of the NKCC1 secretory cotransporter have been isolated from the European eel. The NKCC1a isoform exhibited mRNA expression in a wide range of tissues in a similar fashion to mammals, whereas NKCC1b was expressed primarily in the brain. The effect of freshwater (FW) to seawater (SW) transfer on NKCC1a expression was dependent on the developmental stage. In non-migratory yellow eels, NKCC1a mRNA expression in the gill was transiently up-regulated 4.3-fold after 2 days but also subsequently by 2.5-6-fold 3 weeks after SW transfer. Gill NKCC1a expression was localised mainly in branchial chloride cells of SW acclimated yellow eels. In contrast to yellow eels, NKCC1a mRNA abundance was not significantly different following SW acclimation in silver eel gill. NKCC1a mRNA abundance decreased in the kidney following SW acclimation and this may correlate with lower tubular ion/fluid secretion and urine flow rates in SW teleosts. Kidney NKCC1a mRNA expression in silver eels was also significantly lower than in yellow eels, suggesting some pre-acclimation of mRNA levels. NKCC1a mRNA was expressed at similar low levels in the middle intestine of FW- and SW-acclimated yellow or silver eels, suggesting the presence of an ion secretory mechanism in this gut segment.     

Chronic umbilical cord compression results in accelerated maturation of lung and brown adipose tissue in the sheep fetus during late gestation

Umbilical cord compression (UCC) sufficient to reduce umbilical blood flow by 30% for 3 days, results in increased fetal plasma cortisol and catecholamines that are likely to promote maturation of the fetal lung and brown adipose tissue (BAT). We determined the effect of UCC on the abundance of uncoupling protein (UCP)1 (BAT only) and -2, glucocorticoid receptor (GR), and 11beta-hydroxysteroid dehydrogenase (11beta-HSD)1 and -2 mRNA, and mitochondrial protein voltage-dependent anion channel (VDAC) and cytochrome c in these tissues. At 118 +/- 2 days of gestation (dGA; term approximately 145 days), 14 fetuses were chronically instrumented. Eight fetuses were then subjected to 3 days of UCC from 125 dGA, and the remaining fetuses were sham operated. All fetuses were then exposed to two 1-h episodes of hypoxemia at 130 +/- 1 and 134 +/- 1 dGA before tissue sampling at 137 +/- 2 dGA. In both tissues, UCC upregulated UCP2 and GR mRNA, plus VDAC and cytochrome c mitochondrial proteins. In lung, UCC increased 11beta-HSD1 mRNA but decreased 11beta-HSD2 mRNA abundance, a pattern reversed for BAT. UCC increased UCP1 mRNA and its translated protein in BAT. UCP2, GR, 11beta-HSD1 and -2 mRNA, plus VDAC and cytochrome c protein abundance were all significantly correlated with fetal plasma cortisol and catecholamine levels, but not thyroid hormone concentrations, in the lung and BAT of UCC fetuses. In conclusion, chronic UCC results in precocious maturation of the fetal lung and BAT mitochondria, an adaptation largely mediated by the surge in fetal plasma cortisol and catecholamines that accompanies UCC.