Mechanistic characterization of gastric copper transport in rainbow trout.

An in vitro gut-sac technique and (64)Cu as a radiotracer were used to characterize gastric copper (Cu) transport. Cu transport was stimulated by low luminal pH (4.0 vs. 7.4), to a greater extent than explained by the increased availability of the free Cu(2+) ion. At pH = 4.0, uptake kinetics were indicative of a low affinity (K (m) = 525 µmol L(-1)), saturable carrier-mediated component superimposed on a large linear (diffusive and/or convective) component, with about 50% occurring by each pathway at Cu = 50 µmol L(-1). Osmotic gradient experiments showed that solvent drag via fluid transport may play a role in Cu uptake via the stomach, in contrast to the intestine. Also unlike the intestine, neither the Na(+) gradient, high Ag, nor phenamil had any influence on gastric Cu transport, and a tenfold excess of Fe and Zn failed to inhibit Cu uptake. These findings indicate that neither Na(+)-dependent pathways nor DMT1 are likely candidates for carrier-mediated Cu transport in the stomach. We have cloned a partial cDNA sequence for the copper transporter Ctr1, and show its mRNA expression in all segments of the trout gastrointestinal tract, including the stomach. Based on the fact that this transporter is functional at low pH conventionally found in the stomach lumen, we suggest Ctr1 is a pathway for gastric Cu transport in trout. Extreme hypoxia inhibited Cu uptake. High P(CO2) levels (7.5 torr) increased Cu uptake and acetazolamide (100 µmol L(-1)) significantly inhibited Cu uptake, indicating carbonic anhydrase activity was involved in gastric Cu transport. Transport of Cu was insensitive to bafilomycin (10 µmol L(-1)) suggesting a V-ATPase did not play a direct role in the process. Expression (mRNA) of H (+) , K (+)-ATPase, carbonic anhydrase 2, and the α-3 isoform of Na (+)-K (+)-ATPase were observed in the stomach. We suggest these enzymes facilitate Cu transport in the stomach indirectly as part of a physiological mechanism exporting H(+) to the cell exterior. However, pre-treatment with the H (+) , K (+)-ATPase proton pump blocker omeprazole did not affect gastric Cu transport, suggesting that other mechanisms must also be involved.

Rhesus glycoprotein and urea transporter genes are expressed in early stages of development of rainbow trout (Oncorhynchus mykiss).

The objective of this study was to determine if the genes for the putative ammonia transporters, Rhesus glycoproteins (Rh) and the facilitated urea transporter (UT) were expressed during early development of rainbow trout, Oncorhynchus mykiss Walbaum. We predicted that the Rh isoforms Rhbg, Rhcg1 and Rhcg2 would be expressed shortly after fertilization but UT expression would be delayed based on the ontogenic pattern of nitrogen excretion. Embryos were collected 3, 14 and 21 days postfertilization (dpf), whereas yolk sac larvae were sampled at 31 dpf and juveniles at 60 dpf (complete yolk absorption). mRNA levels were quantified using quantitative polymerase chain reaction and expressed relative to the control gene, elongation factor 1alpha. All four genes (Rhbg, Rhcg1, Rhcg2, UT) were detected before hatching (25-30 dpf). As predicted, the mRNA levels of the Rh genes, especially Rhcg2, were relatively high early in embryonic development (14 and 21 dpf), but UT mRNA levels remained low until after hatching (31 and 60 dpf). These findings are consistent with the pattern of nitrogen excretion in early stages of trout development. We propose that early expression of Rh genes is critical for the elimination of potentially toxic ammonia from the encapsulated embryo, whereas retention of the comparatively benign urea molecule until after hatch is less problematic for developing tissues and organ systems.

Ammonia excretion in rainbow trout (Oncorhynchus mykiss): evidence for Rh glycoprotein and H+-ATPase involvement.

Branchial ammonia transport in freshwater teleosts is not well understood. Most studies conclude that NH(3) diffuses out of the gill and becomes protonated to NH(4)(+) in an acidified gill boundary layer. Rhesus (Rh) proteins are new members of the ammonia transporter superfamily and rainbow trout possess genes encoding for Rh30-like1 and Rhcg2. We identified seven additional full-length trout Rh cDNA sequences: one Rhag and two each of Rhbg, Rhcg1, and Rh30-like. The mRNA expression of Rhbg, Rhcg1, and Rhcg2 was examined in trout tissues (blood, brain, eye, gill, heart, intestine, kidney, liver, muscle, skin, spleen) exposed to high external ammonia (HEA; 1.5 mmol/l NH(4)HCO(3), pH 7.95, 15 degrees C). Rhbg was expressed in all tissues, Rhcg1 was expressed in brain, gill, liver, and skin, and Rhcg2 was expressed in gill and skin. Brain Rhbg and Rhcg1 were downregulated, blood Rh30-like and Rhag were downregulated, and skin Rhbg and Rhcg2 were upregulated with HEA. After an initial uptake of ammonia into the fish during HEA, excretion was reestablished, coinciding with upregulations of gill Rh mRNA in the pavement cell fraction: Rhcg2 at 12 and 48 h, and Rhbg at 48 h. NHE2 expression remained unchanged, but upregulated H(+)-ATPase (V-type, B-subunit) and downregulated carbonic anhydrase (CA2) expression and activity were noted in the gill and again expression changes occurred in pavement cells, and not in mitochondria-rich cells. Together, these results indicate Rh glycoprotein involvement in ammonia transport and excretion in the rainbow trout while underscoring the significance of gill boundary layer acidification by H(+)-ATPase.

The comparative physiology of food deprivation: from feast to famine.

The ability of animals to survive food deprivation is clearly of considerable survival value. Unsurprisingly, therefore, all animals exhibit adaptive biochemical and physiological responses to the lack of food. Many animals inhabit environments in which food availability fluctuates or encounters with appropriate food items are rare and unpredictable; these species offer interesting opportunities to study physiological adaptations to fasting and starvation. When deprived of food, animals employ various behavioral, physiological, and structural responses to reduce metabolism, which prolongs the period in which energy reserves can cover metabolism. Such behavioral responses can include a reduction in spontaneous activity and a lowering in body temperature, although in later stages of food deprivation in which starvation commences, activity may increase as food-searching is activated. In most animals, the gastrointestinal tract undergoes marked atrophy when digestive processes are curtailed; this structural response and others seem particularly pronounced in species that normally feed at intermittent intervals. Such animals, however, must be able to restore digestive functions soon after feeding, and these transitions appear to occur at low metabolic costs.