Stephen D. McCormick

Endocrine Control of Osmoregulation in Teleost Fish

SYNOPSIS. As the primary link between environmental change and physiological response, the neuroendocrine system is a critical part of osmoregulatory adaptations. Cortisol has been viewed as ‘the’ seawater-adapting hormone in fish and prolactin as ‘the’ fresh water adapting hormone. Recent evidence indicates that the growth hormone/insulin-like growth factor I axis is also important in seawater adaptation in several teleosts of widely differing evolutionary lineages. In salmonids, growth hormone acts in synergy with cortisol to increase seawater tolerance, at least partly through the upregulation of gill cortisol receptors. Cortisol under some conditions may promote ion uptake and interacts with prolactin during acclimation to fresh water. The osmoregulatory actions of growth hormone and prolactin are antagonistic. In some species, thyroid hormones support the action of growth hormone and cortisol in promoting seawater acclimation. Although a broad generalization that holds for all teleosts is unlikely, our current understanding indicates that growth hormone promotes acclimation to seawater, prolactin promotes acclimation to fresh water, and cortisol interacts with both of these hormones thus having a dual osmoregulatory function.

11 Hormonal Control of Gill Na+,K+-ATPase and Chloride Cell Function

Publisher Summary This chapter examines the hormonal control of gill Na + , K + -ATPase and chloride cell function. The secretion of excess sodium and chloride by teleosts in seawater is carried out by gill chloride cells. These highly specialized cells are characterized by a large, columnar appearance, numerous mitochondria, an extensive tubular system, an apical crypt, and mucosal–serosal exposure. The breakdown of ATP is detected in the presence and absence of ouabain. Herndon found that prolactin treatment of seawater-adapted tilapia resulted in a dramatic reduction in chloride cell size without changing chloride cell density. Chloride cell height and the proportion of cells spanning the opercular membrane were reduced, suggesting that these cells were effectively removed as chloride secretory cells. Based on reductions in opercular membrane conductance and short-circuit current following prolactin the treatment of seawater-adapted tilapia, Foskett postulated that prolactin reduces chloride cell numbers and active transport in the remaining chloride cells. Working with the closely related but stenohaline Nile tilapia ( Oreochromis niloticus ), Pisam found that prolactin injection caused the appearance of the smaller β-chloride cells that were previously absent from these fish in seawater.

Differential expression of gill Na + ,K + -ATPase - and -subunits, Na + ,K + ,2Cl - cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar

SUMMARY This study examines changes in gill Na+,K+-ATPase (NKA) α- and β-subunit isoforms, Na+,K+,2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR I and II) in anadromous and landlocked strains of Atlantic salmon during parr-smolt transformation, and after seawater (SW) transfer in May/June. Gill NKA activity increased from February through April, May and June among both strains in freshwater (FW), with peak enzyme activity in the landlocked salmon being 50% below that of the anadromous fish in May and June. Gill NKA-α1b, -α3, -β1 and NKCC mRNA levels in anadromous salmon increased transiently, reaching peak levels in smolts in April/May, whereas no similar smolt-related upregulation of these transcripts occurred in juvenile landlocked salmon. Gill NKA-α1a mRNA decreased significantly in anadromous salmon from February through June, whereas α1a levels in landlocked salmon, after an initial decrease in April, remained significantly higher than those of the anadromous smolts in May and June. Following SW transfer, gill NKA-α1b and NKCC mRNA increased in both strains, whereas NKA-α1a decreased. Both strains exhibited a transient increase in gill NKA α-protein abundance, with peak levels in May. Gill α-protein abundance was lower in SW than corresponding FW values in June. Gill NKCC protein abundance increased transiently in anadromous fish, with peak levels in May, whereas a slight increase was observed in landlocked salmon in May, increasing to peak levels in June. Gill CFTR I mRNA levels increased significantly from February to April in both strains, followed by a slight, though not significant increase in May and June. CFTR I mRNA levels were significantly lower in landlocked than anadromous salmon in April/June. Gill CFTR II mRNA levels did not change significantly in either strain. Our findings demonstrates that differential expression of gill NKA-α1a, -α1b and -α3 isoforms may be important for potential functional differences in NKA, both during preparatory development and during salinity adjustments in salmon. Furthermore, landlocked salmon have lost some of the unique preparatory upregulation of gill NKA, NKCC and, to some extent, CFTR anion channel associated with the development of hypo-osmoregulatory ability in anadromous salmon.

Atlantic salmon smolts are more responsive to an acute handling and confinement stress than parr

Abstract Atlantic salmon parr and smolts reared under a natural temperature and photoperiod regime were subjected to an acute handling and confinement stress in early May. Smolts had a mean plasma cortisol concentration of 10 ng/ml before stress and 242 ng/ml 3 h after initiation of stress which returned to pre-stress levels within 8 h. Parr had a plasma cortisol concentration of 4 ng/ml prior to stress which increased to 11 ng/ml 3 h after initiation of stress and returned to pre-stress levels within 8 h. Plasma glucose was significantly higher in parr and smolts 3 h after initiation of stress; in parr, plasma glucose returned to pre-stress levels within 8 h, but not until 48 h in smolts. Plasma chloride concentration in smolts decreased from 139 to 124 mM 3 h after initiation of stress but returned to pre-stress levels within 24 h; plasma chloride in parr was not altered by stress. Plasma thyroxine of parr and smolts peaked at 3 h after initiation of stress and returned to pre-stress levels within 8 h, but smolts had 72% higher levels at 3 h. Pre-smolts (February) and smolts (May) reared under constant temperature (8–10°C) were also subjected to a handling and confinement stress. Although peak levels of plasma cortisol 3 h after initiation of stress were twice as high in smolts, other physiological and endocrine responses were not substantially different between pre-smolts and smolts. The results demonstrate that Atlantic salmon smolts are more responsive to stress than parr and that developmental differences are more important than seasonal changes.

Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (Oreochromis mossambicus) embryos, by means of triple immunofluorescence staining for Na+/K+-ATPase, Na+/K+/2Cl- cotransporter and CFTR anion channel.

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.

Distinct freshwater and seawater isoforms of Na+/K+-ATPase in gill chloride cells of Atlantic salmon

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.

Stimulation of coho salmon growth by insulin-like growth factor I ☆

The effect of insulin-like growth factor I on growth rate of coho salmon (Oncorhynchus kisutch) was examined. Juvenile coho salmon received implants of osmotic minipumps containing recombinant bovine insulin-like growth factor I (rbIGF-I) or saline for a period of 3 to 4 weeks. High doses of rbIGF-I (greater than 0.13 microgram.g-1.d-1) resulted in hypoglycemia and death. In 2-year-old coho salmon, 0.09 microgram.g-1.d-1 rbIGF-I administered for 25 days doubled linear growth rate and increased growth rate in weight by 40%. In rapidly growing, 1-year-old coho salmon, growth rate was not altered by rbIGF-I at 0.01 or 0.05 micrograms.g-1.d-1 for 31 days. In ration-limited fish exhibiting slow growth in the control group, rbIGF-I (0.02 microgram.g-1.d-1) increased linear growth rate by up to threefold and growth rate in weight by up to fourfold. The results indicate that exogenous treatment with mammalian IGF-I can stimulate coho salmon growth under some conditions, and that endogenous IGF-I may be an important factor in regulating growth of teleosts.

Are we missing a mineralocorticoid in teleost fish? Effects of cortisol, deoxycorticosterone and aldosterone on osmoregulation, gill Na + ,K + -ATPase activity and isoform mRNA levels in Atlantic salmon

It has long been held that cortisol, acting through a single receptor, carries out both glucocorticoid and mineralocorticoid actions in teleost fish. The recent finding that fish express a gene with high sequence similarity to the mammalian mineralocorticoid receptor (MR) suggests the possibility that a hormone other than cortisol carries out some mineralocorticoid functions in fish. To test for this possibility, we examined the effect of in vivo cortisol, 11-deoxycorticosterone (DOC) and aldosterone on salinity tolerance, gill Na(+),K(+)-ATPase (NKA) activity and mRNA levels of NKA alpha 1a and alpha 1b in Atlantic salmon. Cortisol treatment for 6-14 days resulted in increased, physiological levels of cortisol, increased gill NKA activity and improved salinity tolerance (lower plasma chloride after a 24h seawater challenge), whereas DOC and aldosterone had no effect on either NKA activity or salinity tolerance. NKA alpha 1a and alpha 1b mRNA levels, which increase in response to fresh water and seawater acclimation, respectively, were both upregulated by cortisol, whereas DOC and aldosterone were without effect. Cortisol, DOC and aldosterone had no effect on gill glucocorticoid receptor GR1, GR2 and MR mRNA levels, although there was some indication of possible upregulation of GR1 by cortisol (p=0.07). The putative GR blocker RU486 inhibited cortisol-induced increases in salinity tolerance, NKA activity and NKA alpha 1a and alpha 1b transcription, whereas the putative MR blocker spironolactone had no effect. The results provide support that cortisol, and not DOC or aldosterone, is involved in regulating the mineralocorticoid functions of ion uptake and salt secretion in teleost fish.

Environmental endocrinology of salmon smoltification.

Smolting is a hormone-driven developmental process that is adaptive for downstream migration and ocean survival and growth in anadromous salmonids. Smolting includes increased salinity tolerance, increased metabolism, downstream migratory and schooling behavior, silvering and darkened fin margins, and olfactory imprinting. These changes are promoted by growth hormone, insulin-like growth factor I, cortisol, thyroid hormones, whereas prolactin is inhibitory. Photoperiod and temperature are critical environmental cues for smolt development, and their relative importance will be critical in determining responses to future climate change. Most of our knowledge of the environmental control and endocrine mediation of smolting is based on laboratory and hatchery studies, yet there is emerging information on fish in the wild that indicates substantial differences. Such differences may arise from differences in environmental stimuli in artificial rearing environments, and may be critical to ocean survival and population sustainability. Endocrine disruptors, acidification and other contaminants can perturb smolt development, resulting in poor survival after seawater entry.

Variation in salinity tolerance, gill Na+/K+-ATPase, Na+/K+/2Cl- cotransporter and mitochondria-rich cell distribution in three salmonids Salvelinus namaycush, Salvelinus fontinalis and Salmo salar.

SUMMARY We compared seawater tolerance, gill Na+/K+-ATPase and Na+/K+/2Cl– cotransporter (NKCC) abundance, and mitochondria-rich cell (MRC) morphology of three salmonids, lake trout Salvelinus namaycush, brook trout Salvelinus fontinalis and Atlantic salmon Salmo salar. They were transferred directly from 0 p.p.t. (parts per thousand; freshwater) to 30 p.p.t. seawater, or transferred gradually from 0 to 10, 20 and 30 p.p.t. at 1-week intervals and kept in 30 p.p.t. for 3 weeks. The survival rates of lake trout, brook trout and Atlantic salmon were 80%, 50% and 100% following direct transfer, and 80%, 100% and 100% during gradual transfer, respectively. Plasma Na+, K+ and Cl– concentrations in surviving lake trout increased rapidly and remained at high levels in 30 p.p.t. of both direct and gradual transfer, whereas those in brook trout showed a transient increase following direct transfer but did not change significantly during gradual transfer. Only minor changes in plasma ions were observed in Atlantic salmon smolts in both direct and gradual transfer. These results suggest that lake trout retains some degree of euryhalinity and that brook trout possesses intermediate euryhalinity between lake trout and Atlantic salmon smolts. Gill Na+/K+-ATPase activity of lake trout and brook trout increased in seawater, whereas that of Atlantic salmon smolts was already upregulated in freshwater and remained high after seawater exposure. NKCC abundance was upregulated in parallel with gill Na+/K+-ATPase activity in each species. Immunocytochemistry with anti-Na+/K+-ATPaseα -subunit and anti-NKCC revealed that the two ion transporters were colocalized on the basolateral membrane of gill MRCs. Immunopositive MRCs were distributed on both primary filaments and secondary lamellae in all three species kept in freshwater; following transfer to seawater this pattern did not change in lake trout and brook trout but lamellar MRCs disappeared in Atlantic salmon. Previous studies on several teleost species have suggested that filament and lamellar MRCs would be involved in seawater and freshwater acclimation, respectively. However, our results in lake trout and brook trout suggest that lamellar MRCs could be also functional during seawater acclimation.