Cytochrome c oxidase is regulated by modulations in protein expression and mitochondrial membrane phospholipid composition in estivating African lungfish.

We examined some of the potential mechanisms lungfish (Protopterus dolloi) use to regulate cytochrome c oxidase (CCO), during metabolic depression. CCO activity was reduced by 67% in isolated liver mitochondria of estivating fish. This was likely accomplished, in part, by the 46% reduction in CCO subunit I protein expression in the liver. No change in the mRNA expression levels of CCO subunits I, II, III, and IV were found in the liver, suggesting CCO is under translational regulation; however, in the kidney, messenger limitation may be a factor as the expression of subunits I and II were depressed ( approximately 10-fold) during estivation, suggesting tissue-specific mechanisms of regulation. CCO is influenced by mitochondrial membrane phospholipids, particularly cardiolipin (CL). In P. dolloi, the phospholipid composition of the liver mitochondrial membrane changed during estivation, with a approximately 2.3-fold reduction in the amount of CL. Significant positive correlations were found between CCO activity and the amount of CL and phosphatidylethanolamine within the mitochondrial membrane. It appears CCO activity is regulated through multiple mechanisms in P. dolloi, and individual subunits of CCO are regulated independently, and in a tissue-specific manner. It is proposed that altering the amount of CL within the mitochondrial membrane may be a means of regulating CCO activity during metabolical depression in the African lungfish, P. dolloi.

Failure to up-regulate gill Na+,K+-ATPase alpha-subunit isoform alpha1b may limit seawater tolerance of land-locked Arctic char (Salvelinus alpinus).

Many populations of Arctic char (Salvelinus alpinus) are land-locked, physically separated from the ocean by natural barriers and unable to migrate to sea like anadromous populations. Previous studies which experimentally transferred land-locked Arctic char to seawater report high mortality rates due to osmoregulatory failure and an inability to up-regulate gill Na(+),K(+)-ATPase activity. This study examined the mRNA expression of two recently discovered alpha-subunit isoforms of gill Na(+)K(+)-ATPase (alpha1a and alpha1b) during seawater exposure of land-locked Arctic char. mRNA levels of these gill Na(+),K(+)-ATPasealpha-subunit isoforms were compared to Na(+),K(+)-ATPase activity and protein levels and related to osmoregulatory performance. Land-locked Arctic char were unable to regulate plasma osmolality following seawater exposure. Seawater exposure did not induce an increase in gill Na(+),K(+)-ATPase activity or protein levels. Na(+),K(+)-ATPase isoform alpha1a mRNA quickly decreased upon exposure to seawater, while isoform alpha1b levels were unchanged. These results suggest the inability of land-locked Arctic char to acclimate to seawater is due a failure to up-regulate gill Na(+),K(+)-ATPase activity which may be due to their inability to increase Na(+),K(+)-ATPase alpha1b mRNA expression.

Intermediary metabolism of Arctic char Salvelinus alpinus during short-term salinity exposure.

The migration of Arctic char Salvelinus alpinus from freshwater to seawater requires a substantial reorganization of the osmoregulatory tissues to regulate plasma ion levels. These modifications have an inherent metabolic cost, which must be met through the upregulation of intermediary metabolism. Arctic char intermediary metabolism was monitored during the initial 96 h of seawater acclimation through measurement of key enzymes in gill, liver, red and white muscle as well as tissue and blood free amino acid (FAA) levels, and plasma glucose and non-esterified fatty acid content. In general, seawater exposure stimulated large changes in amino acid metabolism, but no change in lipid or carbohydrate metabolism. White muscle FAA content increased significantly following seawater exposure, with levels of essential FAAs doubling after 96 h. Similar increases were seen in the plasma, suggesting a rapid mobilization of FAAs to the circulation. These changes were accompanied by significant increases in the activities of enzymes involved in amino acid metabolism in the gill, liver, red and white muscle, suggesting seawater-acclimated fish have an enhanced capacity for energy production from amino acids. Increased energy requirements were evident in the gill of seawater-acclimated char, as citrate synthase activity increased significantly. The results of this study suggest a rapid upregulation of amino acid metabolism may be critical for the successful acclimation of Arctic char to seawater.

Wild Arctic char (Salvelinus alpinus) upregulate gill Na+,K+-ATPase during freshwater migration.

The successful acclimation of eurhyhaline fishes from seawater to freshwater requires the gills to stop actively secreting ions and start actively absorbing ions. Gill Na(+),K(+)-ATPase is known to be an integral part of the active ion secretion model of marine fishes, but its importance in the active ion uptake model of freshwater fishes is less clear. This study, conducted in the high Arctic, examines gill Na(+),K(+)-ATPase regulation in wild anadromous arctic char returning to freshwater from the ocean. Gill Na(+),K(+)-ATPase activity, protein expression, and mRNA expression of Na(+),K(+)-ATPase isoforms alpha 1a and alpha 1b were monitored in arctic char at three points along their migration route to and from Somerset Island, Nunavut, Canada: out at sea (Whaler's Point), in seawater near the river mouth (Nat's Camp), and after entering the Union River. Arctic char collected from the Union River had more than twofold greater gill Na(+),K(+)-ATPase activity. This was associated with a significant increase (threefold) in Na(+),K(+)-ATPase isoform alpha 1a mRNA expression and a significant increase in plasma sodium and osmolality levels compared with seawater char. Compared with char sampled from Whaler's Point, Na(+),K(+)-ATPase isoform alpha 1b mRNA expression was decreased by approximately 50% in char sampled at Nat's Camp and the Union River. These results suggest that the upregulation of gill Na(+),K(+)-ATPase activity is involved in freshwater acclimation of arctic char and implicate a role for Na(+),K(+)-ATPase isoform alpha 1a in this process. In addition, we discuss evidence that arctic char go through a preparatory phase, or "reverse smoltification," before entering freshwater.

Nitrogen metabolism and excretion in the mangrove killifish Rivulus marmoratus I. The influence of environmental salinity and external ammonia.

At a field site in Belize, mangrove killifish Rivulus marmoratus inhabit hypersaline waters (up to 48 per thousand containing approximately 1 mmol l(-1) ammonia. We tested the hypotheses that R. marmoratus modify their nitrogen metabolism and excretion (i) by accumulating free amino acids (FAAs) and urea in the tissues during hyperosmotic stress and (ii) by shifting to ureotelism and accumulating FAAs during hyperammonia stress. Urea excretion (J(Urea)) (but not ammonia excretion, J(Amm)) displayed a diurnal pattern, with significantly less (75 %) urea excreted at night than during the day in both laboratory-reared clones and wild-caught killifish. When fish were exposed to hypersaline conditions (45 per thousand sea water), J(Urea) was significantly reduced and tissue urea and FAA levels were elevated compared with those of control fish (15 per thousand sea water). When R. marmoratus were exposed to 0, 1, 2, 5 and 10 mmol l(-1) NH(4)Cl (pH 8) for 48 h, no differences were found in J(Urea). Remarkably, prolonged exposure (10 days) to 5 mmol l(-1) NH(4)Cl (pH 8) did not result in an elevation of tissue ammonia levels. In addition, tissue urea and total FAA levels did not differ between control and ammonia-exposed fish after > or =4 days. We propose that the euryhaline R. marmoratus retain urea and FAAs within their tissues in response to extreme osmotic stress. In contrast to many ammonia-tolerant fishes, R. marmoratus do not shift to ureotelism during prolonged hyperammonia stress, nor do they convert nitrogenous wastes into FAAs. The data suggest that killifish continue to eliminate ammonia despite an unfavourable blood-to-water gradient, thereby avoiding accumulation of ammonia.

Nitrogen metabolism and excretion in the mangrove killifish Rivulus marmoratus II. Significant ammonia volatilization in a teleost during air-exposure.

The mangrove killifish Rivulus marmoratus can tolerate prolonged periods of air-exposure (>1 month). During these periods of emersion, we hypothesized that R. marmoratus would convert potentially toxic ammonia into urea and free amino acids (FAAs). In air-exposed fish, both ammonia (J(Amm)) and urea (J(Urea)) excretion continued at approximately 57 % and 39 %, respectively, of submerged rates. Remarkably, approximately 42 % of the total ammonia excreted during air-exposure was through NH(3) volatilization. Ammonia did not accumulate in whole-body tissues of air-exposed fish, but levels of both urea and some FAAs (primarily alanine and glutamine) were up to twofold higher after 10 days. The activities of the ornithine-urea cycle enzymes carbamoyl phosphate synthetase III and ornithine transcarbamylase increased (by approximately 30 % and 36 %, respectively) in whole-body tissues of air-exposed fish, while levels of arginase remained unchanged. The activities of enzymes involved in amino acid and oxidative metabolism were not significantly different between control and air-exposed fish. Partitioning of the anterior and posterior ends of immersed fish revealed that just over half (57 %) of the total nitrogen (ammonia+urea) was excreted through the anterior end of the fish, presumably via the branchial tissues, while emersed fish increased excretion via the posterior end (kidney+skin). R. marmoratus do not undergo a shift towards ureotelism during air-exposure. Rather, we propose that R. marmoratus are able to survive on land for extended periods without significant ammonia accumulation because they continuously release ammonia, partially by NH(3) volatilization.