GABA-ergic control of prolactin release in rainbow trout (Oncorhynchus mykiss) pituitaries in vitro

The involvement of γ-aminobutyric acid (GABA) in the control of prolactin (PRL) release was investigated in rainbow trout using both perifused pituitary fragments and pituitary cells in primary culture. In our perifusion system, infusion of GABA (10⁻⁶ to 10⁻⁴ M) caused an inhibition of PRL release (between 20 and 40%). Administration on perifused pituitary fragments of 3APS, a GABAa agonist, mimicked this inhibitory effect. Moreover, bicuculline, a specific antagonist of GABAa receptors, totally abolished GABA effect. When tested on cultured pituitary cells during 40h exposure, GABA (10⁻⁵ M) caused a significant decrease in PRL release (24.5%). Baclofen, a specific agonist for GABAb receptor tested at 10⁻⁶ and 10⁻⁵ M, also inhibited PRL released from cultured pituitary cells. These results demonstrate that GABA inhibits PRL release by acting directly on pituitary cells and that probably both types of GABA receptor (a and b) are involved in this regulation.

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Résumé Nous avons étudié l'implication de l'acide γ-aminobutirique (GABA) dans le controle de la sécrétion de prolactine (PRL) chez la truite arc-en-ciel en utilisant à la fois des fragments d'hypophyse perifusés et des cellules hypophysaires en culture. Dans notre système de périfusion, le GABA (10⁻⁶ à 10⁻⁴ M) inhibe la libération de PRL (entre 20 et 40%). L'administration sur les fragments d'hypophyse périfusés de 3APS, un agoniste des récepteurs GABAa, reproduit ces effets inhibiteurs. De plus, la bicuculline, un antagoniste spécifique des récepteurs de type GABAa, abolie complètement les effets du GABA. Lorsqu'il est testé pendant 40h sur des cellules en culture, le GABA (10⁻⁵ M) réduit de manière significative la libération de PRL (24.5%). Le Baclofen, un agoniste spécifique des récepteurs GABAb testé à 10⁻⁶ et 10⁻⁵ M, inhibe aussi la libération de PRL par les cellules en culture. Ces résultats démontrent que le GABA inhibe la libération de PRL en agissant directement sur les cellules hypophysaires et que les 2 types de récepteurs GABA (a et b) sont impliqués dans cette régulation.

     

Control of gallbladder motility in the rainbow trout,Salmo gairdneri

Immunohistochemistry revealed nerves containing VIP-like and 5-HT-like material in both gallbladder wall and bile duct of the rainbow trout, while endocrine cells containing gastrin/CCK-like and substance P-like material were present in the mucosa of the bile duct and the duodenum. Fluorescence histochemistry showed adrenergic nerves close to the muscle layer of the gallbladder.Sulphated CCK8, caerulein and non-sulphated CCK8 (in this order of potency), 5-HT and acetylcholine were excitatory on isolated strip preparations, while VIP and adrenergic agonists were inhibitory. The adrenergic drugs were probably actingvia a beta-adrenergic receptor, while the effects of 5-HT and cholinergic drugs were antagonized by methysergide and atropine respectively.Electrical stimulation of the gallbladder nervesin situ failed to show any effect or under certain conditions induced a rebound effect.It is concluded that the motility control of the rainbow trout gallbladder may involve an inhibitory innervation by adrenergic and possibly VIP-releasing nerves, while 5-HT, acetylcholine and a CCK-like substance may be involved in the excitatory control.     

Induction of metallothionein synthesis in rainbow trout,Salmo gairdneri,during long-term exposure to waterborne cadmium

Rainbow trout were exposed to 200 g cadmium/l in the water during four months at 6–10°C. The liver, kidney and gills were analyzed for cadmium, copper, zinc, metallothionein and metallothionein mRNA. Cadmium accumulated in all three organs and reached the highest concentration in the kidney. The tissue zine and copper concentrations showed no major alterations during the experiment. The cytosolic distribution of cadmium, copper and zinc was followed during four months of exposure by Sephadex G-75 chromatography. It was found that cadmium was predominantly associated with proteins of an apparent molecular vieght of 10,000 daltons. These proteins were further identified as metallothioneins after fast protein liquid chromatography on a Mono-Q column. The metallothionein concentration was significantly higher in liver of exposed fish than in control fish after only one month. The kidneys reached significantly elevated levels of metallothionein in the exposed group after three months. In the gills, elevated metallothionein concentrations were observed after four months of exposure. After four months of exposure, the metallothionein mRNA content of liver and kidney was analyzed using a rainbow trout anti-sense RNA probe. Elevated MT mRNA levels were observed in both kidney and liver. These results demonstrate thatde novo synthesis of metallothionein is induced by cadmium in rainbow trout after exposure to the metalvia water.     

The liver parenchymal cells of rainbow trout (Salmo gairdneri) endocytose mannose-terminated glycoproteins

A mannose-terminated glycoprotein,¹²⁵I-invertase, was taken up and degraded by isolated rainbow trout liver cells at 12°C. The uptake was inhibited by EGTA and no degradation occurred in the presence of ammonium ions. The liver cell suspension was fractionated by differential centrifugation in parenchymal and nonparenchymal cells, respectively. The parenchymal liver cells seemed to be the most active cells in uptake of labelled invertase bothin vitro andin vivo. Only negligible amounts of ligand were recovered in the nonparenchymal cells. Internalization of¹²⁵I-invertase at different temperatures was demonstrated indirectly by releasing surface-bound ligand with EGTA. Ligand was internalized even at 0°C in trout liver cells.In vitro uptake of¹²⁵I-invertase was inhibited by excess unlabelled invertase, by mannan and by N-acetylglucosamine.These data suggest that invertase is endocytosed by a mannose-specific pathway by the parenchymal liver cells of rainbow trout.     

Structural characterization of the trisaccharide chain from the skin mucus glycoprotein of the rainbow trout, Salmo gairdneri

Skin mucus glycoprotein (RGP) was extracted from the skin mucus of rainbow trout, Salmo gairdneri. Alkaline borohydride treatment of RGP yielded a major oligosaccharide alditol NeuAcα2 to6GalNAc-ol and several minor oligosaccharide alditols. A trisaccharide alditol (OS-I) was purified by Sephadex G-25 gel filtration and high-performance liquid chromatography (HPLC). The amount of OS-I was estimated to be 7% in RGP from the amount of GalNAc-ol. Structural determination of the OS-I was examined. Structure of OS-I was identified to be NeuAcα2 to6 (GalNAcα1 to3) GalNAc-ol by α-N-acetylgalactosaminidase digestion, methylation analysis, and ¹H-NMR spectroscopy. This revised version was published online in August 2006 with corrections to the Cover Date.     

Dietary carbohydrate and growth,body composition and heat increment in rainbow trout (Salmo gairdneri)

A study was conducted to determine growth, body composition and heat increment (HI) of rainbow trout reared on isonitrogenous high digestible carbohydrate (HC) and high fat (HL) diets at 15°C. Trout reared on the HC diet had a significantly lower final body weight and carcass lipid content and a significantly higher feed:gain ratio and carcass protein content than trout reared on the HL diet after 12 weeks on the test diets. The lower carcass lipid composition indicates that trout do not readily convert dietary carbohydrates into fat. HI of trout reared on the HC diet was significantly higher than that of fish reared on the HL diet. Considering the poor utilization of dietary carbohydrates as an energy source and the apparently low conversion of dietary glucose into fat, the cause of the higher HI associated with the HC diet is not completely understood. However, it is possible the elimination of excess dietary glucose is an energy dependent process resulting in an increased heat production which would partly account for the increased HI. Furthermore, if the HC diet is a low net energy diet, then the metabolism and utilization of dietary and tissue proteins (amino acids) for energy by the trout may also be partly responsible for the increased HI of the trout reared on the HC diet.     

Oxidation of glutathione during hydroperoxide metabolism in isolated hepatocytes of rainbow trout (Salmo gairdneri)

Freshly isolated rainbow trout hepatocytes were exposed to tert-butyl hydroperoxide (BuOOH), a substrate for glutathione peroxidase. BuOOH at a concentration approximately equimolar (1 mM) with intracellular reduced glutathione (GSH) caused a reversible increase in intracellular glutathione disulphide (GSSG) but did not compromise cell viability or damage membrane lipids. BuOOH at 10 mM caused a large irreversible increase in intracellular GSSG followed by efflux into the medium. Considerable leakage of lactate dehydrogenase and loss of highly unsaturated fatty acids, particularly docosahexaenoic acid also occurred. Dependence of hydroperoxide removal on flux through the hexose monophosphate pathway was suggested by the increased release of ¹⁴CO₂ from [1-¹⁴C] glucose from hepatocytes incubated with BuOOH.     

Thyroidal compensation in rainbow trout (Salmo gairdneri) fed canola meal

Rainbow trout (Salmo gairdneri Richardson) were fed either a soybean mealbased (SM) or canola meal-based (CM) diet for up to 20 weeks. Plasma thyroxine (T₄) and triiodothryonine (T₃) levels were significantly lower in the CM-fed fish sampled after 12 weeks. However, there appeared to be some compensation after 12 and 20 weeks in that the thyroid hormone levels in trout fed the CM were not significantly different from those of the SM-fed fish. Nevertheless, there was marked thyroid hyperplasia and hypertrophy in the CM-fed fish sampled at 12, 16 and 20 weeks after commencement of the experiment. Moreover, the growth rate was significantly lower in the CM-fed fish in comparison to the SM-fed fish throughout the 20 week study period.Plasma T₄ levels were similar in SM-fed fish sampled 12, 16 and 20 weeks after commencement of the experiment, but plasma T₃ levels progressively increased over this period, as did the apparent activity of the thyroid tissue based on histological criteria.Fasting for up to 8 weeks resulted in the arrested growth of the SM-fed fish, and a loss in body weight of the CM-fed animals over the 8 week period of the fast. In addition, the plasma thyroid hormone levels in the fasted fish tended to be lower than in fish fed both the SM and CM diets prior to fasting, and there was histological evidence indicating a reduced activity of the pituitary-thyroid axis. However, thyroid hyperplasia and hypertrophy were still evident in the fasted fish previously fed the CM diet indicating that the adverse affects of CM diets are not completely reversible after 8 weeks.In fish fed the CM diet for 12 weeks and then the SM diet for up to a further 8 weeks (diet C-S) there was a compensatory increase in plasma thyroid hormone levels evident within 4 weeks after the change in diet, but no apparent decrease in thyroid hyperplasia or hypertrophy. In addition, in the fish fed the C-S diet there was a marked compensatory growth rate, and an increased feed: gain ratio; body weights of this group of fish were not significantly different from those of the SM-fed animals after 20 weeks of study, indicating a considerably higher growth rate over the last 8 week period.     

The effect of size on the mechanical properties of the myotomal-skeletal system of rainbow trout (Salmo gairdneri)

The length and mean cross-sectional area of the myotome of rainbow trout,Salmo gairdneri, scale isometrically with total length (L, cm) and L² respectively for fish from 5 to 35 cm in length. The net maximum force, (F, kN·m^–2) developed by a single twitch of thein situ myotome on one side of the body, and measured normal to the hypural complex increased as; F=1.05×10^–3·L^2.6, and maximum lateral velocity (W, m·s^–1) at the hypural plate as; W=0.29 L^–0.47. Maximum power (P, W) increased as; P=7.64×10^–5·L^3.06. Acceleration rates predicted from these data do not agree well with observations. In addition, except for small fish, predicted maximum speeds differed from those calculated from minimum twitch times of excised muscle blocks and stride length, the popular method for estimating maximum speed. It is suggested that temporal summation of twitches must occur in larger fish. This could provide forces matched to inertial loads which are important in fitness-critical maneuvers.     

Relationship between transmembrane potential and activation of motility in rainbow trout (Salmo gairdneri)

A hypothesis is developed that activation of motility in rainbow trout spermatozoa is a result of membrane hyperpolarization. This hypothesis was developed to explain experimental observations of a relationship between membrane potential and motility as revealed by the use of voltage sensitive fluorescent dyes. The results lead to the following conclusions: a) Transmembrane potential hyperpolarizes with decreasing KCl concentration in 100 mM NaCl. b) Transmembrane potential hyperpolarizes with decreasing NaCl concentration. c) NaCl is three time less effective in changing transmembrane potential and two orders of magnitude less effective in inhibiting activation of motility than KCl. d) Chloride ions have little effect on transmembrane potential or motility. e) Increases in osmotic pressure with the non-ionic molecule sucrose increased the amount of KCl required to inhibit activation. f) The major effect of Na⁺ on K⁺ inhibition may be osmotic.It is suggested that while sperm cells are in the seminal plasma in the reproductive tract of the male rainbow trout their transmembrane potential is maintained above an activation threshold, probably through Na/K pumps which are found in almost all animal cells. Since K⁺ is the most important ion in determining the transmembrane potential, hyperpolarization of the plasma membrane below an activation threshold occurs when the sperm cells are diluted, during spawning, into the low K⁺ environment of freshwater.     

Variations in hepatic metallothionen,zinc and copper levels during an annual reproductive cycle in rainbow trout,Salmo gairdneri

The normal variations in hepatic levels of metallothionein, zinc and copper were studied during an annual reproductive cycle in rainbow trout of both sexes. In female fish, the total hepatic zinc levels closely followed the estradiol-17 and the LSI levels. Hence, the zinc levels rose in September, peaked in December and dropped in January. No distinct peaks were, however, observed in the whole-liver copper content. The hepatic metallothionein levels in female fish began to increase at the onset of exogenous vitellogenesis. Maximum levels were reached after estradiol-17 and LSI levels had dropped in January. In male fish no distinct peaks in either zinc or copper levels were observed. The metallothionein levels increased somewhat during the time of spermatogenesis. It is suggested that metallothionein may regulate the hepatic zinc distribution during the annual reproductive cycle in female rainbow trout, thereby ensuring the organism of a control mechanism to keep the pool of available zinc at an appropriate level.     

Competitive exclusion of brown trout Salmo trutta L., by rainbow trout Oncorhynchus mykiss Walbaum, in lake tributaries, New Zealand

Spawning runs of brown trout Salmo trutta L., and rainbow trout, Oncorhynchus mykiss Walbaum, were compared in three tributaries of two lakes for timing, numbers and redd characteristics. Redd characteristics for the two species in four other streams were examined. No significant differences in redd characteristics between the two species were observed, but in all lake tributaries the possibilities of superimposition of spring spawning rainbow trout on redds of brown trout were examined. A common feature was the high ratio of numbers of spawners to the area of preferred spawning gravel, and it was concluded that in the lake tributaries, superimposition resulted in increased mortality of brown trout eggs, and in one case elimination of the brown trout population. Management possibilities for control of the species proportions in comparable situations are discussed.     

Cytochrome P-450-dependent metabolism in isolated liver cells from fed and starved rainbow trout,Salmo gairdneri

The cytochrome P-450 content in liver cells from rainbow trout was not affected by starvation for 12 weeks whereas the rate of cytochrome P-450-dependent deethylation of 7-ethoxycoumarin in liver cells from 6 or 12 weeks starved fish was 60% of the rate in fed fish. Treatment of fish with -naphthoflavone increased the 7-ethoxycoumarin metabolism several-fold in both starved and fed fish.Optimal cytochrome P-450 monooxygenation in liver cells from fed or starved fish was not affected by addition of glucose or 2-bromooctanoate, an inhibitor of fatty acid -oxidation which is the main source of metabolic fuel in trout liver. The cellular content of NADPH, an obligatory cofactor for cytochrome P-450 monooxygenation, was not affected by addition of substrate to cytochrome P-450, inhibition of fatty acid -oxidation or inhibition of the oxidative phosphorylation. This indicates a great capacity of rainbow trout liver cells to retain high NADPH/NADP⁺ ratios. These results suggest that the cytochrome P-450 mediated metabolism of xenobiotics in liver cells from fed or starved trout is not limited by the availability of reducing equivalents.     

Endocytosis via the scavenger- and the mannose-receptor in rainbow trout (Salmo gairdneri) pronephros is carried out by nonphagocytic cells

Endocytosis of modified human serum albumin (HSA) and mannose-terminated glycoproteins was studied in pronephros cells from rainbow trout (Salmo gairdneri). Blood clearance and tissue uptake of dinitrophenylated human serum albumin (DNP-HSA) was dependent on the number of DNP-groups conjugated to the albumin molecule. Uptake of DNP₃₅-HSA in isolated pronephros cells was saturable. Pronephros cells also internalized the mannose-terminated glycoprotein invertase by a receptor-mediated process. DNP-HSA and invertase were recovered in the same cell fractions when pronephros cells containing in vivo endocytosed ligands were separated by density gradient centrifugation. The cells endocytosing these ligands were apparently not macrophages. The macrophages were recovered in cell fractions with higher densities. They were identified by their ability to adhere to glass and to carry out phagocytosis. Cultured macrophages did not endocytose chemically modified albumin (DNP-HSA and formaldehyde-treated albumin) and mannose-terminated glycoproteins (ovalbumin) in vitro. These ligands were not recovered in glass adherent pronephros cells after in vivo endocytosis of the proteins. The present data suggest that macrophages in rainbow trout pronephros possess neither the receptor for chemically modified albumin, the scavenger receptor, nor the mannose-specific receptor.     

Effect of excess iodide on thyroid function of rainbow trout,Salmo gairdneri

The acute and chronic effects of excess iodide (KI or NaI) were studied on thyroid function of rainbow trout at 11±1°C. No Wolff-Chaikoff effect, characteristic of mammals, was observed and instead plasma L-thyroxine (T₄) levels increased 6 hr after a single iodide injection. Plasma 3,5,3′-triiodo-L-thyronine (T₃) did not change and by 24 hr plasma T₄ returned to normal. This iodide-induced elevation in plasma T₄ was probably not due to toxic effects demonstrated at higher NaI or KI doses. A single iodide injection also decreased the plasma iodide distribution space, decreased the fractional rate of plasma iodide loss and completely blocked thyroidal uptake of radioiodide. Injections of iodide over a 22-day period elevated plasma iodide 200X with no mortality and no influence on plasma T₄ or T₃. It is concluded that: (i) apart from the transient 6h increase in plasma T₄, trout thyroid function, as judged by plasma hormone levels, is insensitive to considerable iodide excess, (ii) non-invasive iodide suppression of thyroidal radioiodide recycling may be useful in kinetic studies of¹²⁵I-labeled thyroid hormones, and (iii) fundamental differences in intrathyroidal iodine metabolism appear to exist between mammals and fish.     

Response of resident brown trout, Salmo trutta L., and rainbow trout, Oncorhynchus mykiss (Walbaum), to the stocking of hatchery-reared brown trout

Two strains of hatchery-reared adult brown trout, Salmo trutta L., [208–334 mm total length (TL); n =  591] were individually marked and released into a limestone stream. The estimated survival after one month (86%; n =  508) was comparable to that for resident brown trout and rainbow trout, Oncorhynchus mykiss (Walbaum), (89%; n =  771), but declined to 14% ( n =  83) after 8 months compared with 52% ( n =  451) for resident trout. The movement of resident trout out of stocked stretches was higher (14%) than from control sites (5%), but the population size in both individual sites and the overall study area were unaffected. The growth of resident brown trout was unaffected by stocking, but rainbow trout showed lower growth rates in stocked versus unstocked stretches both one and 8 months after stocking ( P <  0.002).     

Does oral 3,5,3 ′-triiodo-l-thyronine affect dietary glucose utilization and plasma insulin levels in rainbow trout (Salmo gairdneri)?

A factorial experiment was conducted to determine the effect and interaction of dietary carbohydrate level and triiodo-L-thyronine (T₃) supplementation on the growth, physiological response and plasma insulin and cortisol levels of rainbow trout. The oral administration of T₃ significantly increased the growth, protein efficiency ratio and feed efficiency of trout, indicating an increased protein and perhaps energy utilization in these fish. However, T, administration did not significantly increase the utilization of dietary glucose as an energy source by the trout. Similarly, the administration of T₃ did not significantly affect plasma insulin levels in either the fed or the fasted trout. Plasma insulin levels were significantly higher in fed trout reared on the non-T₃ supplemented high carbohydrate diet in comparison to trout reared on the low carbohydrate diets. This indicates that increased dietary carbohydrate stimulates increased insulin secretion in the trout. Therefore, although rainbow trout are not insulin-deficient, they can still be considered a diabetic-like animal due to their poor glucose tolerance. Plasma cortisol levels were not affected by diet composition and altered plasma glucose levels.     

The dynamics of vitellogenin sequestration into vitellogenic ovarian follicles of the rainbow trout,Salmo gairdneri

This study provides quantitative data on the dynamics of protein sequestration into vitellogenic follicles of the rainbow trout, Salmo gairdneri. The ovarian uptake of both radiolabelled vitellogenin (VTG) and bovine serum albumin (BSA) were investigated in a homogenous population of maturing vitellogenic females. Ten fish were injected with ³H. VTG directly into the bloodstream. Concomitantly, five of these fish received an equal amount of ¹⁴C.BSA. Twenty two hours after injection, of the tissues sampled, the greatest proportions of ³H. VTG were present in the blood (into which the radio labels were administered) and in the ovary (up to 28% and 46% of that originally present in the blood, respectively). VTG uptake was both selective, rates of uptake far exceeding that of the ¹⁴C.BSA, and rapid. ³H. VTG was sequestered at rates of between 35 to 390 ng.mm² follicle surface⁻¹.h⁻¹ in the different fish. The rates of VTG uptake into similarly sized follicles varied both between different sites within the ovary (by up to 30%) and also between the ovaries (by up to 38%) of an individual fish.     

Circulatory and ionoregulatory effects of atrial natriuretic peptide on rainbow trout (Salmo gairdneri Richardson) fed normal or high levels of dietary salt

Rainbow trout fed a normal salt diet (1.3% NaCl) or a high salt diet (12% NaCl for at least 6 months) were chronically cannulated in the dorsal aorta and received 10 μg kg⁻¹ ANP (1–28 human, UBC-Bioproducts) infused over a 10 min period. This had an insignificant influence on sodium balance, blood electrolytes and branchial sodium fluxes. In fish given a normal diet, the blood pressure and heart rate were uninfluenced by ANP, but pulse pressure was reduced by on average 60% and in some cases was not evident at all. Blood pressure in the fish fed a high salt diet was significantly higher than in the control fish; this together with heart rate and pulse pressure was not affected by ANP administration.