Social status affects lipid metabolism in rainbow trout, Oncorhynchus mykiss.

Juvenile rainbow trout ( Oncorhynchus mykiss) confined in pairs form social hierarchies in which socially subordinate fish display characteristic traits, including reduced growth rates and altered glucose metabolism. These effects are, in part, mediated by chronically elevated cortisol levels and/or reduced feeding. To determine the effects of social status on lipid metabolism, trout were held in pairs for 4 days, following which organismal and liver-specific indexes of lipid metabolism were measured. At the organismal level, circulating triglycerides were elevated in dominant trout, whereas subordinate trout exhibited elevated concentrations of circulating free fatty acids (FFAs) and lowered plasma total cholesterol levels. At the molecular level, increased expression of lipogenic genes in dominant trout and cpt1a in subordinate trout was identified, suggesting a contribution of increased de novo lipogenesis to circulating triglycerides in dominant trout and reliance on circulating FFAs for ß-oxidation in the liver of subordinates. Given the emerging importance of microRNAs (miRNA) in the regulation of hepatic lipid metabolism, candidate miRNAs were profiled, revealing increased expression of the lipogenic miRNA-33 in dominant fish. Because the Akt-TOR-S6-signaling pathway is an important upstream regulator of hepatic lipid metabolism, its signaling activity was quantified. However, the only difference detected among groups was a strong increase in S6 phosphorylation in subordinate trout. In general, the changes observed in lipid metabolism of subordinates were not mimicked by either cortisol treatment or fasting alone, indicating the existence of specific, emergent effects of subordinate social status itself on this fuel.

Molecular cloning and characterization of secretory carbonic anhydrase VI in pufferfish (Takifugu rubripes).

Carbonic anhydrase VI (CA VI) has been characterized as a secretory isozyme in mammals. Our present study confirmed the occurrence of CA VI in pufferfish (Takifugu rubripes). In this study, genomic sequence information for the CA VI of pufferfish was used for molecular cloning. We cloned a 1821 bp cDNA sequence, which consisted of a complete coding sequence of 1623bp and a deduced amino acid sequence of 540 amino acids from the open reading frame. A BLAST search indicated that this protein exhibits 53%, 79%, and 67% identity with human, tilapia, and gar CA VI, respectively. It also shows 63%-77% identity with other fish CA VI-like sequences (zebrafish, Asian arowana, salmon, and large yellow croaker). Moreover, alignment of two or more sequences revealed that the protein sequence of pufferfish CA VI has 34%-37% identity with mammalian and fish CA II sequences. An NH2-terminal signal peptide of 18 amino acids in length was predicted in the pufferfish CA VI sequence. Three potential N-linked glycosylation sites and two cysteine residues (Cys-28 and Cys-209) that are likely to form one disulfide bond were present in pufferfish CA VI. In silico and phylogenetic analyses revealed that pufferfish CA VI is an extracellular secretory protein. Active site analysis indicated that this protein is a low-activity CA isozymes due to a characteristic Val/Ile substitution at position 207. Homology modeling of puffer CA VI was performed using the crystal structure of human carbonic anhydrase XIV as a template structure, based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and in situ hybridization results revealed that, the pufferfish CA VI is highly expressed in liver tissue.

Air breathing and aquatic gas exchange during hypoxia in armoured catfish.

Air breathing in fish is commonly believed to have arisen as an adaptation to aquatic hypoxia. The effectiveness of air breathing for tissue O2 supply depends on the ability to avoid O2 loss as oxygenated blood from the air-breathing organ passes through the gills. Here, we evaluated whether the armoured catfish (Hypostomus aff. pyreneusi)-a facultative air breather-can avoid branchial O2 loss while air breathing in aquatic hypoxia, and we measured various other respiratory and metabolic traits important for O2 supply and utilization. Fish were instrumented with opercular catheters to measure the O2 tension (PO2) of expired water, and air breathing and aquatic respiration were measured during progressive stepwise hypoxia in the water. Armoured catfish exhibited relatively low rates of O2 consumption and gill ventilation, and gill ventilation increased in hypoxia due primarily to increases in ventilatory stroke volume. Armoured catfish began air breathing at a water PO2 of 2.5 kPa, and both air-breathing frequency and hypoxia tolerance (as reflected by PO2 at loss of equilibrium, LOE) was greater in individuals with a larger body mass. Branchial O2 loss, as reflected by higher PO2 in expired than in inspired water, was observed in a minority (4/11) of individuals as water PO2 approached that at LOE. Armoured catfish also exhibited a gill morphology characterized by short filaments bearing short fused lamellae, large interlamellar cell masses, low surface area, and a thick epithelium that increased water-to-blood diffusion distance. Armoured catfish had a relatively low blood-O2 binding affinity when sampled in normoxia (P50 of 3.1 kPa at pH 7.4), but were able to rapidly increase binding affinity during progressive hypoxia exposure (to a P50 of 1.8 kPa). Armoured catfish also had low activities of several metabolic enzymes in white muscle, liver, and brain. Therefore, low rates of metabolism and gill ventilation, and a reduction in branchial gas-exchange capacity, may help minimize branchial O2 loss in armoured catfish while air breathing in aquatic hypoxia.

Cortisol-sensitive urea transport across the gill basolateral membrane of the gulf toadfish (Opsanus beta).

Gulf toadfish (Opsanus beta) use a unique pulsatile urea excretion mechanism that allows urea to be voided in large pulses via the periodic insertion or activation of a branchial urea transporter. The precise cellular and subcellular location of the facilitated diffusion mechanism(s) remains unclear. An in vitro basolateral membrane vesicle (BLMV) preparation was used to test the hypothesis that urea movement across the gill basolateral membrane occurs through a cortisol-sensitive carrier-mediated mechanism. Toadfish BLMVs demonstrated two components of urea uptake: a linear element at high external urea concentrations, and a phloretin-sensitive saturable constituent (K(m) = 0.24 mmol/l; V(max) = 6.95 micromol x mg protein(-1) x h(-1)) at low urea concentrations (<1 mmol/l). BLMV urea transport in toadfish was unaffected by in vitro treatment with ouabain, N-ethylmaleimide, or the absence of sodium, conditions that are known to inhibit sodium-coupled and proton-coupled urea transport in vertebrates. Transport kinetics were temperature sensitive with a Q(10) > 2, further suggestive of carrier-mediated processes. Our data provide evidence that a basolateral urea facilitated transporter accelerates the movement of urea between the plasma and gills to enable the pulsatile excretion of urea. Furthermore, in vivo infusion of cortisol caused a significant 4.3-fold reduction in BLMV urea transport capacity in lab-crowded fish, suggesting that cortisol inhibits the recruitment of urea transporters to the basolateral membrane, which may ultimately affect the size of the urea pulse event in gulf toadfish.

Mechanisms behind Pb-induced disruption of Na+ and Cl- balance in rainbow trout (Oncorhynchus mykiss).

The mechanism of Pb-induced disruption of Na(+) and Cl(-) balance was investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Na(+) and Cl(-) influx rates were reduced immediately in the presence of 2.40 +/- 0.24 and 1.25 +/- 0.14 muM Pb, with a small increase in efflux rates occurring after 24-h exposure. Waterborne Pb caused a significant decrease in the maximal rate of Na(+) influx without a change in transporter affinity, suggesting a noncompetitive disruption of Na(+) uptake by Pb. Phenamil and bafilomycin markedly reduced Na(+) influx rate but did not affect Pb accumulation at the gill. Time-course analysis in rainbow trout exposed to 0, 0.48, 2.4, and 4.8 microM Pb revealed time- and concentration-dependent branchial Pb accumulation. Na(+)-K(+)-ATPase activity was significantly reduced, with 4.8 microM exposure resulting in immediate enzyme inhibition and 0.48 and 2.4 microM exposures inhibiting activity by 24 h. Reduced activity was weakly correlated with gill Pb accumulation after 3- and 8-h exposures; this relationship strengthened by 24 h. Reduced Na(+) uptake was correlated with gill Pb burden after exposures of 3, 8, and 24 h. Immediate inhibition of branchial carbonic anhydrase activity occurred after 3-h exposure to 0.82 +/- 0.05 or 4.30 +/- 0.05 microM Pb and continued for up to 24 h. We conclude that Pb-induced disruption of Na(+) and Cl(-) homeostasis is in part a result of rapid inhibition of carbonic anhydrase activity and of binding of Pb with Na(+)-K(+)-ATPase, causing noncompetitive inhibition of Na(+) and Cl(-) influx.

Time course analysis of the mechanism by which silver inhibits active Na+ and Cl- uptake in gills of rainbow trout.

A time course analysis using (110m)Ag, (24)Na(+), and (36)Cl(-) examined gill silver accumulation and the mechanism by which waterborne silver (4.0 x 10(-8) M; 4.3 microg/l) inhibits Na(+) and Cl(-) uptake in gills of freshwater rainbow trout. Analyses of gill and body fluxes allowed calculation of apical uptake and basolateral export rates for silver, Na(+), and Cl(-). To avoid changes in silver bioavailability, flow-through conditions were used to limit the buildup of organic matter in the exposure water. For both Na(+) and Cl(-) uptake, apical entry, rather than basolateral export, was the rate-limiting step; Na(+) and Cl(-) uptake declined simultaneously and equally initially, with both uptakes reduced by approximately 500 nmol.g(-1).h(-1) over the 1st h of silver exposure. There was a further progressive decline in Na(+) uptake until 24 h. Carbonic anhydrase activity was inhibited by 1 h, whereas Na(+)-K(+)-ATPase activity was not significantly inhibited until 24 h of exposure. These results indicate that carbonic anhydrase inhibition can explain the early decline in Na(+) and Cl(-) uptake, whereas the later decline is probably related to Na(+)-K(+)-ATPase blockade. Contrary to previous reports, gill silver accumulation increased steadily to a plateau. Despite the rapid inhibition of apical Na(+) and Cl(-) uptake, apical silver uptake (and basolateral export) increased until 10 h, before decreasing thereafter. Thus silver did not inhibit its own apical uptake in the short term. These results suggest that reduced silver bioavailability is the mechanism behind the pattern of peak and decline in gill silver accumulation previously reported for static exposures to silver.

High plasma buffering and the absence of a red blood cell beta-NHE response in brown bullhead (Ameiurus nebulosus).

The high non-bicarbonate buffer capacity of brown bullhead (Ameiurus nebulosus) plasma was postulated to function as an alternative mechanism for the protection of red blood cell (RBC) intracellular pH (pHi) in the absence or attenuation of a RBC adrenergic response. The requirement for protecting RBC pHi arises from the presence of a Root effect haemoglobin in bullhead. In support of this hypothesis, bullhead RBCs incubated in vitro with isoproterenol (10(-8)-10(-5) mol l(-1)) or forskolin (10(-4) mol l(-1)) exhibited significant cyclic AMP accumulation, but failed to exhibit cell swelling or significant Na(+) or Cl(-) accumulation; plasma pH (pHe) was also unaffected. Similarly, no significant effect on RBC water content, Na(+) or Cl(-) concentration, or pHe was detected in bullhead blood incubated with 8-bromo cyclic AMP (10(-4)-10(-2) mol l(-1)) in vitro. These results suggest that while bullhead RBCs possess a beta-adrenoreceptor linked to cyclic AMP formation, stimulation of this adrenergic receptor does not result in measurable activation of a Na(+)/H(+) exchanger.