Ghrelin in the summer flounder: immunolocalization to the gastric glands and action on plasma cortisol levels.

We searched for evidence of the hormone ghrelin in the stomach of a juvenile, marine teleost, the summer flounder. Using antiserum against the conserved core of the ghrelin peptide, immunoreactivity was observed in the simple, branching epithelium that comprises the gastric glands. The immunoreaction was especially strong in the glandular epithelium located deep in the tissue. Next, we assessed a possible connection between ghrelin and the hypothalamic-pituitary-interrenal axis through a series of three injection experiments in which acylated or non-acylated (des-acyl) ghrelin was injected into the peritoneum of summer flounder (Paralichthys dentatus). A significant increase in plasma cortisol relative to saline-injected controls was observed for the acylated form at a dose of 1000 ng g(-1) of body mass in one of the experiments. In another, there was a trend for des-acyl ghrelin at 1000 ng g(-1) of body mass to increase plasma cortisol. Taken together, this study provides evidence that gastric glands in the stomach of summer flounder are a site of ghrelin production and that peripherally administered ghrelin can stimulate the cortisol axis in a teleost.

Evidence for the onset of feedback regulation of cortisol in larval summer flounder.

We investigated the functional development of feedback regulation of cortisol levels during early development in a marine teleost, summer flounder, using a novel pharmaceutical approach. Larvae were immersed for 4h in the glucocorticoid agonist dexamethasone (20 microM) and/or the glucocorticoid-receptor antagonist RU486 (0.12 microM) at 1, 7, or 21 days after hatching. The hypothesis was that, if feedback regulation were operational, tissue cortisol concentrations would be suppressed by dexamethasone and stimulated by RU486. Whole-body cortisol content of 1-day-old larvae was significantly decreased from 0.32 ng/g body weight (mean) to 0.08-0.12 ng/g by immersion in dexamethasone, RU486, or both, perhaps due to displacement of cortisol from the yolk sac. There were no changes in cortisol content among treatment groups in 7-day-old larvae. The expectations of our hypothesis were met in 21-day-old larvae. Immersion in RU486 increased cortisol content from 0.29 ng/g (control) to 2.00 ng/g, whereas immersion in dexamethasone (with or without RU486) suppressed cortisol to 0.03-0.04 ng/g. The results indicate that a fully functional hypothalamic-pituitary-interrenal axis is established by 3 weeks after yolk-sac resorption, but before the onset of metamorphosis in summer flounder. This is the earliest detection of feedback regulation in a teleost fish.

Cortisol is necessary for seawater tolerance in larvae of a marine teleost the summer flounder.

Larval-stage summer flounder (Paralichthys dentatus) were immersed in the corticosteroid-receptor blocker RU486 to test the effects of cortisol deficiency on salinity tolerance. Premetamorphic larvae held at 10 (near isosmotic) or 30 (hyperosmotic) parts per thousand ( per thousand) seawater survived well over 5d in 0, 0.012, or 0.12 microM RU486. However, at concentrations of 1.2 or 3.6 microM RU486, mortality was significantly greater for larvae in 30 per thousand compared to larvae in 10 per thousand. In a separate experiment, the ability of RU486 to inhibit tolerance to hyperosmotic medium (30 per thousand) was confirmed; immersion at 1.2 microM RU486 induced mortality of larvae in the metamorphic climax stage held at 30 per thousand, but not 0 or 10 per thousand. Mortality due to RU486 in pre- or prometamorphic stage larvae was prevented by concurrent immersion in cortisol at concentrations approximately 10-200 times greater than RU486, indicating that the action of RU486 was specific to antagonism of cortisol. The efficacy of 1.2 microM RU486 in reducing survival in 30 per thousand was found to be stage-dependent and exhibited the following hierarchy for fastest time to 50% mortality: prometamorphosis>metamorphic climax>premetamorphosis. In a 5-d pretreatment of pre- or prometamorphic larvae by immersion in 20 microM cortisol and/or 0.12 microM RU486 at 30 per thousand, only RU486 had a limited effect on decreasing survival when larvae were challenged with abrupt exposure to 50 per thousand. In total, the results evidence for the first time a necessary role for cortisol in seawater tolerance of a larval marine teleost.

Adaptation for water balance in the partial gastrointestinal tract of summer flounder.

Marine teleosts continually drink and absorb water across the intestine to prevent dehydration. Surprisingly, summer flounder that are missing most of their intestine, due to necrotizing enteritis, maintain osmotic homeostasis. Here, we tested the hypothesis that this remnant gastrointestinal tract undergoes compensatory adaptation for fluid uptake. Flounder (Paralicthys dentatus) with a partial gastrointestinal tract had an emaciated liver. Moisture content of muscle however was similar to healthy cohorts with an intact gastrointestinal tract, indicative of an undisturbed osmoregulatory status. Mass-specific rates of fluid uptake across all segments of the partial gastrointestinal tract were less than or similar to rates in corresponding segments from intact flounder. In contrast, weights (percent of body mass) were doubled in stomach and partial intestine of the remnant gastrointestinal tract. Consequently, total capacity for fluid uptake (microL h(-1) g body mass(-1)) was similar for both groups. The functional capacity of the remnant gastrointestinal tract was therefore of a magnitude sufficient to maintain osmoregulatory ability, further evidencing a critical role of the intestine in salt and water balance of marine teleosts.

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.

Osmoregulatory physiology of pyloric ceca: regulated and adaptive changes in chinook salmon.

Functions of the anatomically obvious, yet peculiar, pyloric ceca of the fish gut have been a source of conjecture for over two millennia since Aristotle hypothesized on digestive utilities. Here, we demonstrate regulated and adaptive changes in osmoregulatory physiology of ceca from chinook salmon (Onchorhynchus tshawytscha). Transfer of salmon from freshwater to seawater (both short- and long-term) significantly stimulated both fluid uptake from 5.1 to 8.3-9.3 microl/cm2/hr and also Na+/K+ -ATPase from 6.5 to 8.3-9.6 micromol/ADP/mg protein/hr. Similar changes were induced with implants of cortisol, which resulted in high physiological cortisol levels in plasma. Ceca, which can number about 200 in chinook salmon, were estimated to account for the majority of fluid uptake capacity of the intestine and, after long-term seawater adaptation, the proportion of uptake capacity was sixfold higher. Transport physiology of ceca is thus under environmental and endocrine control indicative of an important role in salt and water homeostasis.

Temporal changes in intestinal Na+, K(+)-ATPase activity and in vitro responsiveness to cortisol in juvenile chinook salmon.

Seasonal changes in endogenous Na+, K(+)-ATPase activity were measured in pyloric ceca and posterior intestine of juvenile chinook salmon (Oncorhynchus tshawytscha) maintained in fresh water over 18 months. In tissues from these same fish, the in vitro responsiveness of Na+, K(+)-ATPase activity to 10 microg cortisol/ml was assessed. There were pronounced increases in endogenous Na+, K(+)-ATPase activity in summer for both intestinal regions, in underyearlings and yearlings. In pyloric ceca, a significant positive response of Na+, K(+)-ATPase activity to cortisol, in vitro, was restricted to the months preceding increases in endogenous Na+, K(+)-ATPase and the month afterward. Na+, K(+)-ATPase activity of the posterior intestine was only responsive to cortisol in underyearlings in the period before the peak in endogenous enzyme activity. At a time when explants were responsive to cortisol, in vitro exposure to 0.1-10 microg cortisol/ml resulted in dose-dependent elevations of Na+, K(+)-ATPase activity over controls (0 microg cortisol/ml). The results show that the intestine exhibits increased enzymatic potential for water absorption that is indicative of parr-smolt transformation. Alterations in tissue responsiveness to cortisol may contribute to these changes in Na+, K(+)-ATPase activity of pyloric ceca.

Pseudobranch and gill Na(+), K(+)-ATPase activity in juvenile chinook salmon, Oncorhynchus tshawytscha: developmental changes and effects of growth hormone, cortisol and seawater transfer.

The teleost pseudobranch is a gill-like structure often fused to the anterior of the opercular cavity. Pseudobranch cells are mitochondria rich and have high levels of Na(+), K(+)-ATPase activity. In this study, pseudobranch Na(+), K(+)-ATPase activity in juvenile chinook salmon (Oncorhynchus tshawytscha) was compared to gill Na(+), K(+)-ATPase activity, a known marker of parr-smolt transformation, in three experiments. In two stocks of New Zealand chinook salmon, pseudobranch Na(+), K(+)-ATPase activity was found to significantly increase during development. At these times gill Na(+), K(+)-ATPase activity was also elevated. Pseudobranch Na(+), K(+)-ATPase activity did not increase 10 days after transfer from fresh water to 34 ppt seawater, a treatment that resulted in a twofold increase in gill Na(+), K(+)-ATPase activity. Cortisol (50 microg/g) and ovine growth hormone (5 microg/g) implants had no effect on pseudobranch Na(+), K(+)-ATPase activity in underyearling chinook salmon, while gill Na(+), K(+)-ATPase activity was stimulated by each hormone. In yearling chinook salmon, only cortisol stimulated pseudobranch Na(+), K(+)-ATPase activity 14 days post-implantation. It was concluded that the pseudobranch differs from the gill in terms of the regulation of Na(+), K(+)-ATPase activity and a role during adaptation to seawater is likely to be limited.