Acidosis,glycolysis and energy state in anaerobic heart tissue from rainbow trout

With focus on metabolism not depending on contractility in myocardial tissue from rainbow trout, Oncorhynchus mykiss, the effects of high CO₂ on lactate production, phosphocreatine, creatine, ATP, ADP, AMP and intracellular pH were examined under a blockage of cell respiration either alone or in combination with a glycolytic inhibition. Irrespective of metabolic interventions, a change in CO₂ from 1 to either 11 or 5% of the gas mixture perfusing the muscle bath with 15 mmol·l^-1 HCO _- ³ caused a drop of intracellular pH from 7.4 to either 6.5 or 7.0, respectively. An elevation of CO₂ to 11% diminished the rate of anaerobic lactate formation and slightly lowered anaerobic energy degradation. The further addition of 1 mmol·l^-1 iodoacetate to inhibit glycolysis strongly enhanced the tendency of acidosis to lower energy degradation. Moreover, iodoacetate induced a parallel decrease in ATP and total concentration of phosphorylated adenylates and an increase in resting tension. These effects were all substantially dampened by acidosis and could not immediately be related to tissue content of energy-rich phosphates. Tentatively, the depression of resting tension was the prime effect and a cause of the other effects acidosis. However, these were not affected by an inhibition of resting tension development with 2,3-butadione monoxime. The results suggest that glycolysis protects the anaerobic myocardium also by means not immediately related to tissue energy state. Acidosis exerts a similar protection, which is marginal as long as glycolysis is fully active, but substantial with an inhibited glycolysis.Abbreviations Cr _t total tissue concentration of creatine - G _PCr energy liberated per mol PCr hydrolyzed - IAA iodoacetate - PCr phosphocreatine - PE total tissue concentration of energy-rich phosphate bonds - pH _i intracellular pH - P _i inorganic phosphate - TAN total tissue concentration of phosphorylated adenylates - 2,3-BDM 2,3-butadione monoxime - SE standard error of the mean     

ATP,creatine phosphate,and mechanical activity in rainbow trout myocardium under inhibition of glycolysis and cell respiration

Summary Twitch force and resting tension of electrically stimulated ventricular strips of rainbow trout were compared with tissue contents of phosphocreatine, creatine, and ATP. The phosphocreatine/total creatine ratio, which was used to assess the cytoplasmic phosphorylation potential, fell with the fraction of cell respiration that was inhibited by sodium cyanide and N₂. Concomitantly, twitch force decreased while resting tension tended to increase. This relation between phosphocreatine/total creatine and mechanical parameters became more prominent as glycolysis was increasingly inhibited by sodium iodoacetate. Furthermore, glycolytic inhibition was followed by a decrease in the ATP/phosphocreatine ratio. The latter effect was the same in 1% and 6% CO₂. Thus, it cannot be ascribed to an action of intracellular pH on the creatine kinase catalyzed reaction. Notably, resting tension as well as twitch force relative to ATP was augmented by glycolytic inhibition. The main conclusions are that in the presence of a decreased mitochondrial activity, glycolysis protects contractility not only by counteracting a lowering in high energy phosphates but also by supporting the ATP/phosphocreatine ratio. Apparently, the creatine kinase activity is insufficient to maintain ATP in equilibrium with phosphocreatine. In addition, glycolysis seems to elevate the level of free phosphate relative to ATP, so that twitch force development as well as rigor complex formation is counteracted.     

Isolation and characterization of the receptor for vitellogenin from follicles of the rainbow trout,Oncorhynchus mykiss

The isolation and characterization of the receptor for vitellogenin from follicle membranes of the rainbow trout, Oncorhynchus mykiss, is described. Follicle membrane proteins subjected to SDS-polyacrylamide gel electrophoresis and subsequently to either protein staining or ligand blotting with radiolabelled vitellogenin (¹²⁵iodine-vitellogenin) demonstrated that the vitellogenin receptor has an apparent molecular mass of 200 kD (probably comprising of two 100-kD subunits) under non-reducing conditions. The vitellogenin binding sites were identified as specific receptors: binding was saturable and the binding sites were both tissue specific to follicle membranes and exhibited ligand specificity. Scatchard analyses of specific binding data revealed a single class of binding sites with a high affinity for rainbow trout vitellogenin (K _d=8.2·10^-9 mol·1^-1). Both brown trout, Salmo trutta, vitellogenin and carp, Cyprinus carpio, vitellogenin were able to displace the radiolabelled rainbow trout vitellogenin from its receptor, although they were less effective than rainbow trout vitellogenin.Abbreviations B _max maximum number of binding sites available - BSA bovine serum albumin - bt-VTG brown trout vitellogenin - c-VTG earp vitellogenin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - K _d dissociatian constant - NCM nitrocellulose membranes - PMSF phenylmethylsulphonylfluoride - rt-VTG rainbow trout vitellogenin - VTG vitellogenin     

Thermal adaptation in biological membranes: interacting effects of temperature and pH

Summary The interacting effects of pH and temperature on membrane fluidity were studied in plasma membranes isolated from liver of rainbow trout (Oncorhynchus mykiss) acclimated to 5 and 20°C. Fluidity was determined as a function of temperature under conditions of both constant (in potassium phosphate buffer) and variable pH (in imidazole buffer, consistent with imidazole alphastat regulation) from the fluorescence anisotropy of two probes: 1,6-diphenyl-1,3,5-hexatriene, which intercalates into the bilayer interior, and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene which is anchored at the membrane/water interface. The temperature dependence of the anisotropy parameter for 1,6-diphenyl-1,3,5-hexatriene in plasma membranes of 20°C-acclimated trout was greater when determined in phosphate (AP per °C=-0.047) than in imidazole buffer (AP per °C=-0.022); similar, but less significant, trends were noted with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene. In contrast, the temperature dependence of fluidity (AP/°C in the range-0.0222 to-0.027) did not vary with buffer composition in membranes of 5°C-acclimated trout. In phosphate buffer, anisotropy parameter values for 1,6-diphenyl-1,3,5-hexatriene were significantly lower in 5°C-than 20°C-acclimated trout, indicating a less restricted probe environment following cold acclimation and nearly perfect compensation (91%) of fluidity. Temperature-dependent patterns of acid-base regulation were estimated to account for 11–40% of the fluidization evident in membranes of 5°C-trout, but a period of cold acclimation was required for complete fluidity compensation. In contrast, no homeoviscous adaptation was evident in imidazole buffer, indicating that membrane fluidity is sensitive to buffer composition. Accordingly, vesicles of bovine brain phosphatidylcholine, suspensions of triolein, and plasma membranes of 5°C-acclimated trout were consistently more fluid in imidazole than phosphate buffer. Membranes of 5°C-acclimated trout were enriched in molecular species of phosphatidylcholine containing 22:6n3 (at the expense of species containing 18:1n9 and 18:2n6) compared to membranes of 20°C-trout; consequently, the unsaturation index was significantly higher (3.29 versus 2.73) in trout maintained at 5 as opposed to 20°C. It is concluded that: 1) the chemical composition of the internal milieu can significantly influence the physical properties of membrane lipids; 2) temperature-dependent patterns of intracellular pH regulation may partially offset the ordering effect of low temperature on membrane fluidity in 20°C-acclimated trout transferred to 5°C, but not in 5°C-acclimated trout transferred to warmer temperatures; 3) the majority of the thermal compensation of plasma membrane fluidity resulting from a period of temperature acclimation most likely reflects differences in membrane composition between acclimation groups; 4) imidazole apparently interacts with trout hepatocyte plasma membranes in a unique way.Abbreviations _im netcharge stateofproteins - AP anisotropyparameter - bw body weight - DPH 1,6-diphenyl-1,3,5-hexatriene - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonicacid - PC phosphatidylcholine - pH_e pHofarterial blood - pH_i intracellular pH - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene - TRIS tris(hydroxymethyl)aminomethane     

Identification of four ovarian receptor proteins that bind vitellogenin but not other homologous plasma lipoproteins in the rainbow trout,Oncorhynchus mykiss

Membrane proteins from ovarian follicles, testis and somatic tissues of rainbow trout, Oncorhynchus mykiss, were extracted by ultracentrifugation, separated on sodium dodecyl sulphate gels and isolated on polyvinyl difluoride membranes. Vitellogenin receptor proteins were visualised using protein staining and hybridisation with ¹²⁵I-vitellogenin Four follicle-membrane proteins, with molecular masses of 220, 210, 110 and 100 kDa, showed a strong affinity for vitellogenin and were specific to the ovary. Other homologous lipoproteins (very low density lipoprotein, low density lipoprotein and high density lipoprotein) had a very limited ability to displace ¹²⁵I-vitellogenin from its receptor, indicating that the ovarian receptor proteins were fairly specific for vitellogenin. Proteins with an affinity for very low density lipoprotein and low density lipoprotein were visualised in liver, spleen and muscle, eluting on sodium dodecyl sulphate gels with molecular masses of about 150 kDa. Peptides generated from trypsin digests of the receptor proteins with a high affinity for vitellogenin showed sequence homology with receptors in the lipoprotein family, including a sequence that is believed to act as the internalisation signal [Phe-Asp-Asn-Phe-Tyr-] and, a sequence identity with the recently characterised chicken vitellogenin/very low density lipoprotein receptor [Ser-Glu-Leu-Tyr-Glu-Pro-Ala-]. Together, the ligand blotting and peptide sequence data support the contention that the four ovarian membrane proteins isolated are receptor proteins specific for vitellogenin and they do not bind other plasma lipoproteins to any significant degree.Abbreviations BSA bovine serum albumin - HDL high density lipoprotein - LDL low-density lipoprotein - HPLC high performance liquid chromatograph - PVDF polyvinylidene difluoride - RIA radioimmunoassay - rt-VTG rainbow trout vitellogenin - SDS sodium dodecyl sulphate - VLDL very low density lipoprotein - VTG vitellogenin - VRP-1,-2,-3 or -4 vitellogenin receptor proteins     

Modulation of catecholamine storage and release by the pituitary-interrenal axis in the rainbow trout,Oncorhynchus mykiss

This study examined the effects of pituitary-interrenal hormones on catecholamine storage and release in the rainbow trout Oncorhynchus mykiss. An extract of trout pituitary elicited the release of adrenaline, but not noradrenaline, using an in situ perfusion preparation. A variety of doses of adrenocorticotropic hormone (2–2000 mU) caused the release of both catecholamines in situ which was unaffected by pre-treatment with the ganglion blocker, hexamethonium, or the serotonergic receptor antagonist, methysergide, but was abolished in calcium-free media. Intra-arterial injections of adrenocorticotrophic hormone in vivo caused an elevation of plasma adrenaline but not noradrenaline levels. Injections of cortisol in situ did not elicit catecholamine release. Trout given an intraperitoneal implant of cortisol (50 mg·kg^-1 body weight) had significantly higher plasma cortisol concentrations when compared to controls after 7 days of implantation. Increases in the levels of stored catecholamines were observed in various regions of the kidney and posterior cardinal vein following 3 and 7 days of cortisol treatment. The ability of the chromaffin cells to release catecholamines in response to cholinergic stimulation was assessed in situ after 7 days of treatment. Basal (non-stimulated) adrenaline outflowing perfusate levels were greater in the cortisol-treated fish. Cortisol treatment increased the responsiveness of the catecholamine release process to low doses of the cholinoceptor agonist carbachol. Three or 7 days of cortisol treatment did not alter the in vitro activity of the enzyme phenylethanolamine-N-methyl-transferase. The results of this study demonstrate that interactions within the pituitary-adrenal axis can influence both catecholamine storage and release in the rainbow trout.Abbreviations ACTH adrenocorticotropic hormone - AK anterior third of the kidney - APCV anterior third of the PCV - HPLC high performance liquid chromatography - MK middle third of the kidney - M1 maximum value - MPCV middle third of the PCV - MS222 ethyl-aminobenzoate - P1 pre value - PCA perchloric acid - PCV posterior cardinal vein - PK posterior third of the kidney - PNMT phenylethanolamine-N-methyltransferase - PPCV posterior third of the PCV - rbc red blood cells - SEM standard error of the mean - TK total kidney (i.e. the sum of the AK, MK, and PK) - TPCV total PCV (i.e. the sum of the APCV, MPCV and PPCV)     

Growth hormone stimulates hepatic lipid mobilization in rainbow trout,Oncorhynchus mykiss

Rainbow trout were used to characterize the direct influence of growth hormone on hepatic lipid mobilization in vitro. Liver was removed from fish fasted 72 h, sliced into 1-mm³ pieces and incubated in Hanks' medium containing ovine or salmon growth hormone (0.28 ng·ml^-1–28 g·ml^-1). Lipid mobilization, resulting from triacylglycerol hydrolysis, was evaluated by fatty acid and glycerol release into culture medium. Control rates of fatty acid release and glycerol release were 0.95±0.16 (mean ± SE) and 0.88±0.28 mol·l^-1·mg fresh weight, respectively. Both ovine growth hormone (28 ng·ml^-1) and salmon growth hormone (28 ng·ml^-1) stimulated fatty acid release into culture medium, increasing rates by 112% and 97%, respectively, during the course of a 24-h incubation. Glycerol release was similarly augmented by growth hormone treatment. Growth hormone-stimulated metabolite release became evident within 12 h and persisted for up to 72 h. The presence of amino acids in the culture medium potentiated the lipolytic action of growth hormone. Ovine growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 108% increase in glycerol, release over rates observed in the absence of amino acids. Salmon growth hormone (28 ng·ml^-1) in the presence of amino acids stimulated a 53% increase in fatty acid, and a 44% increase in glycerol, release over rates observed in the absence of amino acids. Ovine growth hormone (28 ng·ml^-1) also stimulated gluconeogenesis, as indicated by a 75% increase in phosphoenolpyruvate carboxykinase activity in liver pieces incubated in the presence of amino acids. These results indicate that growth hormone directly stimulates lipid breakdown in the liver of trout and that amino acids potentiate growth hormone-stimulated lipolysis.Abbreviations AA amino acid(s) - dGDP deoxy-guanosine diphosphate - ED ₅₀ 50% effective dose - FA fatty acid(s) - fw fesh weight - GH growth hormone - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - MS-222 tricaine methanesulfonate - MEM minimum essential medium - oGH ovine growth hormone - PEPCK phosphoenolpyruvate carboxykinase - PK_c protein kinase C - rpm revolutions per minute - sGH salmon growth hormone - TG triacylglycerol - w/v weight per volume This paper was presented in abstract form at the Annual Meeting of the American Society of Zoologists, Dec. 26–30, 1991, Atlanta, GA     

Sarcoplasmic reticulum and excitation-contraction coupling at 20 and 10 °C in rainbow trout myocardium

Summary Isometric force and series membrane potential were recorded in isolated ventricular strips from rainbow trout at 20 and 10 °C. Preparations were electrically stimulated to contract at either 0.5 or 0.2 Hz. Single extrastimulations elicited a twitch force which diminished when the preceding diastole was shortened below the regular value. The stimulation following this extra stimulation evoked no potentiation of force. Apart from a marginal effect on the post extrasystolic force at 20 °C, ryanodine did not affect either of these responses or the steady-state force at 0.5 Hz. At 0.2 Hz the steady-state force was somewhat depressed by ryanodine at 20 but not at 10 °C. In contrast, extrastimulations preceded by diastoles of up to 1 h more than doubled extrasystolic force at 20 °C. This effect was removed by ryanodine. Both the potentiations and the effect of ryanodine were strongly reduced at 10 °C. Apparently, temperature acts on the release of Ca²⁺ from the sarcoplasmic reticulum, since Ca²⁺ seems to be taken up at both temperatures. Hence, at both 20 and 10 °C, the contractures evoked in a solution inhibiting sarcolemmal Ca²⁺ transfer and releasing Ca²⁺ from the sarcoplasmic reticulum were diminished by pretreatment with 15 mM caffeine. Action potential duration at 20 °C was less than half of that at 10 °C. At both temperatures it tended to be prolonged by periods of prolonged rest. No effect of ryanodine on action potential configuration was detected. The results suggest that trout myocardial sarcoplasmic reticulum, although powerful at unphysiologically low stimulation rates, does not partake in the beat-to-beat regulation of force at heart rates encountered in vivo.Abbreviations ESF extrasystolic force - SR sarcoplasmic reticulum - v _F maximal rate of force development - v _R maximal rate of relaxation - TPF time to peak force - TR _0.5 time for half relaxation - TTF duration of force development     

A cDNA microarray assessment of gene expression in the liver of rainbow trout (Oncorhynchus mykiss) in response to a handling and confinement stressor

A purpose-designed microarray platform (Stressgenes, Phase 1) was utilised to investigate the changes in gene expression within the liver of rainbow trout during exposure to a prolonged period of confinement. Tissue and blood samples were collected from trout at intervals up to 648 h after transfer to a standardised confinement stressor, together with matched samples from undisturbed control fish. Plasma ACTH, cortisol, glucose and lactate were analysed to confirm that the neuroendocrine response to confinement was consistent with previous findings and to provide a phenotypic context to assist interpretation of gene expression data. Liver samples for suppression subtractive hybridisation (SSH) library construction were selected from within the experimental groups comprising “early” stress (2–48 h) and “late” stress (96–504 h). In order to reduce redundancy within the four SSH libraries and yield a higher number of unique clones an additional subtraction was carried out. After printing of the arrays a series of 55 hybridisations were executed to cover 6 time points. At 2 h, 6 h, 24 h, 168 h and 504 h 5 individual confined fish and 5 individual control fish were used with control fish only at 0 h. A preliminary list of 314 clones considered differentially regulated over the complete time course was generated by a combination of data analysis approaches and the most significant gene expression changes were found to occur during the 24 h to 168 h time period with a general approach to control levels by 504 h. Few changes in expression were apparent over the first 6 h. The list of genes whose expression was significantly altered comprised predominantly genes belonging to the biological process category (response to stimulus) and one cellular component category (extracellular region) and were dominated by so-called acute phase proteins. Analysis of the gene expression profile in liver tissue during confinement revealed a number of significant clusters. The major patterns comprised genes that were up-regulated at 24 h and beyond, the primary examples being haptoglobin, β-fibrinogen and EST10729. Two representative genes from each of the six k-means clusters were validated by qPCR. Correlations between microarray and qPCR expression patterns were significant for most of the genes tested. qPCR analysis revealed that haptoglobin expression was up-regulated approximately 8-fold at 24 h and over 13-fold by 168 h.     

Parvalbumin expression in trout swimming muscle correlates with relaxation rate

Rainbow trout (Oncorhynchus mykiss) display longitudinal and developmental shifts in muscle relaxation rate. This study aimed to determine the role of variations in parvalbumin content in modulating muscle relaxation. Parvalbumin is a low molecular weight protein that buffers myoplasmic Ca2+ and enhances muscle relaxation. In some fish, longitudinal variations in muscle relaxation have been linked to variations in the total amount of parvalbumin present in muscle and in the relative expression of two parvalbumin isoforms. We have demonstrated previously that anterior slow-twitch or red myotomal muscle relaxes more rapidly than that from the posterior for both rainbow and brook trout. Further, younger rainbow trout parr have faster red muscle relaxation rates than older smolts. Here we report similar results for fast-twitch or white muscle. We quantified the parvalbumin expression in red and white muscle from different body positions of rainbow trout parr and smolts and for brook trout (Salvelinus fontinalis) adults. There was a significant shift in total parvalbumin content of muscle: the faster muscle from the anterior myotome contained greater amounts of parvalbumin. For brook trout, longitudinal variation in relaxation rate was also associated with shifts in the relative expression of the two parvalbumin isoforms. The faster muscle of parr contained more parvalbumin. Lastly, trout white muscle tended to have higher levels of parvalbumin and greater levels of the Parv2 (relative to Parv1) isoform as compared to red muscle. Parvalbumin expression correlated with muscle relaxation rate in trout, although there were species-specific differences in the importance of altering total parvalbumin content versus shifts in relative parvalbumin isoform expression.     

Inhibition of growth hormone synthesis by somatostatin in cultured pituitary of rainbow trout

Summary When the pituitary of rainbow trout (Oncorhynchus mykiss) was incubated in a serum-free medium, a high level of growth hormone release as well as an activation of growth hormone synthesis were observed, suggesting the existence of hypothalamic inhibitory factor(s) on growth hormone synthesis. Although an inhibitory effect of somatostatin on growth hormone release is well established in both mammals and teleosts, an effect on growth hormone synthesis has not been demonstrated. In this study, we examined the effect of somatostatin on growth hormone synthesis in organ-cultured trout pituitary using immunoprecipitation and Northern blot analysis. Somatostatin inhibited growth hormone release from the cultured pituitary within 10 min after addition without affecting prolactin release. Incubation of the pituitary with somatostatin also caused a significant reduction in newly-synthesized growth hormone in a dose-related manner, as assessed by incorporation of [³H]leucine into immunoprecipitable growth hormone. There were no changes in the level or molecular length of growth hormone mRNA after somatostatin treatment, as assessed by Northern slot blot and Northern gel blot analyses. Human growth hormone-releasing factor stimulated growth hormone release, although the spontaneous synthesis of growth hormone was not augmented. However, somatostatin-inhibited growth hormone synthesis was restored by growth hormone-releasing factor to the control level. The spontaneous increase in growth hormone synthesis observed in the organ-cultured trout pituitary may be caused, at least in part, by the removal of the inhibitory effect of hypothalamic somatostatin.Abbreviations GH growth hormone - GHRF GH-releasing factor - PRL prolactin - SDS sodium dodecyl sulphate - SRIF somatostatin (somatropin release-inhibiting factor)     

Use of androgenesis for estimating maternal and mitochondrial genome effects on development and oxygen consumption in rainbow trout, Oncorhynchus mykiss

Chromosome set manipulation was used to produce rainbow trout, Oncorhynchus mykiss, with identical nuclear backgrounds, but different maternal backgrounds to determine mitochondrial effects on development rate and oxygen consumption. Significant differences in development rate and oxygen consumption were observed between groups from different females. Development rates ranged from a mean of 317.97 degree days (°d) to 335.25 °d in progeny from different females. Mean oxygen consumption rates ranged from 3.31 μmol O₂ g^− 1 wet mass h^− 1 to 9.66 μmol O₂ g^− 1 wet mass h^− 1. Oxygen consumption and development rate analysis revealed the two slowest developing groups had the highest oxygen consumption rates. Development rate differences between second generation clonal females indicate that mitochondrial genomes play a significant role on early development and are comparable to development rate differences between clonal lines of rainbow trout. These results indicate that selection for mitochondrial genomes could increase growth rates and possibly food conversion ratios in aquaculture species.     

Effect of salmon gonadotropic hormone on sex steroids in male rainbow trout: plasma levels and testicular secretion in vitro

Summary Male rainbow trout were treated with salmon gonadotropic hormone (GTH) at different stages of the circannual reproductive cycle; spawning fish were also treated with an antiserum against salmon GTH. Injection of GTH led to a several-fold increase of plasma sex steroid levels during spermatogenesis and in the spawning season but was without effect at early stages of testicular development. GTH neutralization during the spawning season was followed by a several-fold decrease of plasma sex steroid levels. During spermatogenesis and in the spawning season, both treatment regimes resulted in an increased sensitivity of testicular explants in response to a subsequent stimulation of steroid secretion in vitro. This up-regulatory response may facilitate and maintain the high sex steroid plasma levels observed during the spawning season. It may also be necessary to allow for concomitant peak values of plasma GTH and sex steroids in the spawning season, a situation difficult to understand within the negative feedback concept. The adaptive capacities of the testicular steroidogenic system indicate that it is not only an effector site for GTH but also an active part of the endocrine system controling reproduction.Abbreviations BSA bovine serum albumin - bw body weight - E₂ 17-estradiol - GnRH gonadotropin releasing-hormone - GTH gonadotropic hormone - LH luteinizing hormone - OHT 11-hydroxytestosterone - OT 11-ketotestosterone - 17-20P 17-hydroxy, 20-dihydroprogesterone - PE pituitary extract - raGTH rabbit anti-GTH antiserum - rPS rabbit preimmune serum - T testosterone     

Changes in expression of provasotocin and proisotocin genes during adaptation to hyper- and hypo-osmotic environments in rainbow trout

Summary The physiological roles of neurohypophysial hormones, vasotocin (VT) and isotocin (IT), are not yet clear in teleosts. Since information on responsiveness of hypothalamic neurosecretory neurons to environmental stimuli may contribute to an understanding of their physiological roles, effects of environmental hyper- and hypo-osmotic stimuli on expression of VT and IT precursor (proVT and proIT) genes in rainbow trout were investigated, using an in situ hybridization technique in which 46 mer synthetic oligonucleotides were used as hybridization probes. The probes corresponded to the mRNA loci encoding chum salmon proVT (-5 to 11) and proIT (-5 to 11), and were labeled at the 3-end with ³⁵S. Autoradiographic silver grains which represent the hybridization signals of proVT and proIT mRNAs were localized in both magnocellular and parvocellular neurons in the nucleus preopticus magnocellularis (NPOmg). Localizations of proVT and proIT hybridization signals coincided with those of VT- and IT-immunoreactive neurons in adjacent sections, and showed that proVT and proIT genes are expressed in separate neurons. The intensity of proVT hybridization signals as determined by grain counting in magnocellular neurons in the NPOmg was conspicuously decreased after transfer from fresh water (FW) to 80% seawater (SW). The proVT mRNA levels in SW trout were consistently lower than those of FW trout for up to 2 weeks. After return from 80% SW to FW, the proVT mRNA level increased, attaining the initial FW level. The proIT mRNA levels in SW trout were not statistically different from those in FW trout, except for the 1st day after transfer to SW. These results suggest that synthesis of proVT was elevated by transfer from higher to lower salinity, and that VT may have a physiological role in salmonid osmoregulation, especially in adaptation to a hypo-osmotic environment.Abbreviations ABC avidin-biotin-peroxidase complex - AVP arginine vasopressin - BSA bovine serum albumin - EDTA ethylene diamine tetra-acetic acid - cpm counts per minute - FW fresh water - GFR gromerular filtration rate - ISH in situ hybridization - IT isotocin - mRNA messenger ribonucleic acid - NPOmg nucleus preopticus magnocellularis - OXT oxytocin - PBS phosphate-buffered saline - SSC saline sodium citrate - RT room temperature - SW seawater - VT vasotocin     

Localization and diurnal variations of carbonic anhydrase mRNA expression in the inner ear of the rainbow trout Oncorhynchus mykiss

Physiological studies have suggested that carbonic anhydrase (CA) plays a central role in otolith biomineralization via ion transport. However, the presence and exact function of CA in the inner ear have not been determined. In the present study, to investigate the localization of CA and its involvement in otolith calcification, we cloned two cDNAs encoding CAs from the rainbow trout sacculus. These two cDNAs, designated rainbow trout CAa (rtCAa) and rtCAb, both had an open reading frame encoding 260 amino acids with a sequence identity of 78%. Remarkably, rtCAb has a high degree of homology (82%) with “high activity CA” in the zebrafish, and its mRNA expression showed variation in the range 1.9–11.4 × 10⁴ copies/ng total RNA in the sacculus. In contrast, rtCAa mRNA was constantly expressed at approximately 3 × 10⁴ copies/ng total RNA. In situ hybridization revealed that rtCAb mRNA was strongly expressed in the distal squamous epithelial cells and transitional epithelial cells, except the mitochondria-rich cells, whereas, rtCAa was localized in extrasaccular tissue. These results suggest that the rtCAb isozyme is involved in the daily increment formation and calcification of otoliths via phase and spatial differences of the bicarbonate supply to the endolymph.     

Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes

This study, using ¹³C nuclear magnetic resonance spectroscopy showed enrichment of glycogen carbon (C1) from ¹³C-labelled (C1) glucose indicating a direct pathway for glycogen synthesis from glucose in rainbow trout (Oncorhynchus mykiss) hepatocytes. There was a direct relationship between hepatocyte glycogen content and total glycogen synthase, total glycogen phosphorylase and glycogen phosphorylase a activities, whereas the relationship was inverse between glycogen content and % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio. Incubation of hepatocytes with glucose (3 or 10 mmol·1^-1) did not modify either glycogen synthase or glycogen phosphorylase activities. Insulin (porcine, 10^-8 mol·1^-1) in the medium significantly decreased total glycogen phosphorylase and glycogen phosphorylase a activities, but had no significant effect on glycogen synthase activities when compared to the controls (absence of insulin). In the presence of 10 mmol·1^-1 glucose, insulin increased % glycogen synthase a and decreased % glycogen phosphorylase a activities in trout hepatocytes. Also, the effect of insulin on the activities of % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio were more pronounced at low than at high hepatocyte glycogen content. The results indicate that in trout hepatocytes both the glycogen synthetic and breakdown pathways are active concurrently in vitro and any subtle alterations in the phosphorylase to synthase ratio may determine the hepatic glycogen content. Insulin plays an important role in the regulation of glycogen metabolism in rainbow trout hepatocytes. The effect of insulin on hepatocyte glycogen content may be under the control of several factors, including plasma glucose concentration and hepatocyte glycogen content.     

Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics

Carnobacterium maltaromaticum B26 and Carnobacterium divergens B33, which were isolated from the intestine of healthy rainbow trout (Oncorhynchus mykiss, Walbaum), were selected as being potentially useful as probiotics with effectiveness against Aeromonas salmonicida and Yersinia ruckeri. Thus, rainbow trout administered with feed supplemented with B26 or B33 dosed at >10(7) cells g(-1) feed conferred protection against challenge with virulent cultures of the pathogens. Moreover, both cultures persisted in the gut for up to 3 weeks after administration. The cultures enhanced the cellular and humoral immune responses. Specifically, fish fed with B26 demonstrated significantly increased phagocytic activity of the head kidney macrophages, whereas the use of B33 led to significant increases in respiratory burst and serum lysozyme activity. Also, the gut mucosal lysozyme activity for fish fed with both cultures was statistically higher than the controls.     

Extinction of a conditioned response in rainbow trout selected for high or low responsiveness to stress

Two lines of rainbow trout (Oncorhynchus mykiss) that exhibit divergent endocrine responsiveness to stressors also display disparate behavioral traits. To investigate whether the high-responding (HR) and low-responding (LR) fish also differ in cognitive function, the rate of extinction of a conditioned response was compared between the two lines. Groups of HR and LR fish were exposed to a paired conditioned stimulus (CS; water off) and unconditioned stimulus (US; confinement stressor). After exposure to 18 CS-US pairings, at least 70% of individuals of both lines acquired a conditioned response (CR) manifested as an elevation of blood cortisol levels on presentation of the CS only. Post-conditioning, the fish were tested by presentation of the CS at weekly intervals, for 4 weeks, with no further reinforcement, and the extinction of the CR in the two lines was compared. The decline in mean plasma cortisol levels after exposure to the CS over successive tests suggested that the CR was retained for a shorter period among the HR (<14 days) than LR fish (<21 days). The frequency of individuals within each line whose plasma cortisol levels indicated a stress response when exposed to the CS was significantly greater among the LR than HR fish at 14 and 21 days with no HR fish falling into this category at 21 days. At 28 days post-conditioning, there were no HR fish and only three LR fish were categorized as "stressed". These results suggest that there are differences in cognitive function between the two lines. Possible mechanisms underlying these differences are discussed.     

Identification and expression analysis of rainbow trout <Emphasis Type="Italic">pumilio</Emphasis>-1 and <Emphasis Type="Italic">pumilio</Emphasis>-2

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.This work was in part supported by a grant from the Akiyama Foundation to E.I. Nucleotide sequence data for rainbow trout pumilio-1 and pumilio-2 have been deposited in the DDBJ/EMBL/GenBank databases.     

Angiotensin II binding to tissues of the rainbow trout,Oncorhynchus mykiss,studied by autoradiography

Summary Tissue slices from seawater-adapted and freshwater-adapted rainbow trout, Oncorhynchus mykiss, were exposed to ¹²⁵I-angiotensin II (1.01·10^-9 M) and binding sites located by light-microscopic autoradiography. Binding/uptake was significantly inhibited by excess (10^-5 M) unlabelled angiotensin II, suggesting specific binding/uptake of angiotensin II to the ventral and dorsal aorta (smooth muscle), urinary bladder (smooth muscle and epithelial lining), glomeruli and proximal tubules, the gill (lamellae and central filament), skin (epithelium), intestine and oesophagus (mucosal epithelium), liver, heart (ventricular myocytes), adrenocortical tissue and brain (cerebellum and medulla oblongata). The specific binding/uptake of angiotensin II to tissues of freshwater- and seawater-adapted animals were generally similar. However, binding/uptake by the proximal tubules was significantly higher in freshwater-adapted trout than seawater-adapted trout. Specific binding/uptake of angiotensin II by the smooth muscle of the bladder was significantly higher in trout adapted to seawater than trout adapted to freshwater.