Elevated levels of trimethylamine oxide in deep-sea fish: evidence for synthesis and intertissue physiological importance

Tissue levels of trimethylamine oxide (TMAO) were compared for seven teleost and two elasmobranch species captured from three depth ranges: shallow (<150 m), moderate (500-700 m), and deep (1,000-1,500 m). Within the teleosts, the deep-caught species had significantly greater TMAO content than shallow- or moderate-caught species. In all teleosts, muscle had substantially more TMAO than all other tissues. Kidney or, in some cases, liver had elevated trimethylamine (TMA) content, 2.20-9.65 mmol/kg, along with appreciable trimethylamine oxidase (TMAoxi) activity, suggesting active TMAO synthesis. No correlation was found between TMAoxi activity and TMAO content. The elasmobranchs in this study, Squalus acanthias and Centroscyllium fabricii from shallow and deep water, respectively, were both squaliform sharks. The deep-caught species had significantly more TMAO in all tissues than the shallow species. Furthermore, urea was significantly less in the deep species in all tissues except liver, while the urea:TMAO ratio was significantly less in all tissues. As with teleosts, the TMAO content of muscle was substantially higher for both elasmobranchs than in all other tissues. TMAoxi was below levels of detection in both elasmobranch species, suggesting that TMAO is obtained solely from the diet. This study expands the trend of increased muscle TMAO in deep-sea fish to a variety of other tissues. The accumulation of TMAO in various tissues in deep-sea teleosts and the accumulation of TMAO and concurrent urea decrease in a deep-sea elasmobranch in comparison to a shallow water species strongly support the contention that TMAO is of physiological importance in deep-sea fish.     

The Physiology and Evolution of Urea Transport in Fishes

This review summarizes what is currently known about urea transporters in fishes in the context of their physiology and evolution within the vertebrates. The existence of urea transporters has been investigated in red blood cells and hepatocytes of fish as well as in renal and branchial cells. Little is known about urea transport in red blood cells and hepatocytes, in fact, urea transporters are not believed to be present in the erythrocytes of elasmobranchs nor in teleost fish. What little physiological evidence there is for urea transport across fish hepatocytes is not supported by molecular evidence and could be explained by other transporters. In contrast, early findings on elasmobranch renal urea transporters were the impetus for research in other organisms. Urea transport in both the elasmobranch kidney and gill functions to retain urea within the animal against a massive concentration gradient with the environment. Information on branchial and renal urea transporters in teleost fish is recent in comparison but in teleosts urea transporters appear to function for excretion and not retention as in elasmobranchs. The presence of urea transporters in fish that produce a copious amount of urea, such as elasmobranchs and ureotelic teleosts, is reasonable. However, the existence of urea transporters in ammoniotelic fish is curious and could likely be due to their ability to manufacture urea early in life as a means to avoid ammonia toxicity. It is believed that the facilitated diffusion urea transporter (UT) gene family has undergone major evolutionary changes, likely in association with the role of urea transport in the evolution of terrestriality in the vertebrates.     

Subcellular location of glutamine synthetase and urea cycle enzymes in liver of spiny dogfish (Squalus acanthias)

Glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, both of which are present in high concentrations in liver of urea-retaining elasmobranchs, have been found to be located exclusively in the mitochondria in liver from the representative elasmobranch Squalus acanthias. This observation is consistent with the view that the function of this unique carbamoyl-phosphate synthetase is related to urea synthesis, and that the initial nitrogen-donating substrate for urea synthesis in these species is glutamine rather than ammonia. The urea cycle enzymes, ornithine carbamoyltransferase and arginase, are also located in the mitochondria, whereas argininosuccinate synthetase and argininosuccinate lyase are located in the cytosol. Glutamine synthetase and arginase are mitochondrial enzymes in uricotelic species, but are normally found in the cytoplasm in ureotelic species. the properties of the elasmobranch arginase, however, are characteristic of arginases from ureotelic species (e.g. the Km for arginine is 1.2 mM, and the enzyme has an Mr congruent to 100,000).     

Purification and properties of glutamine synthetase from liver of Squalus acanthias

Ammonia assimilation for urea synthesis by liver mitochondria in marine elasmobranchs involves, initially, formation of glutamine which is subsequently utilized for mitochondrial carbamoyl phosphate synthesis [P. M. Anderson and C. A. Casey (1984) J. Biol. Chem. 259, 456-462]. The purpose of this study was to determine if the glutamine synthetase catalyzing this first step in urea synthesis has properties uniquely related to this function. Glutamine synthetase has been highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme has a molecular weight of approximately 400,000 in the presence of Mg2+, MgATP, and L-glutamate, but dissociates reversibly to a species with a molecular weight of approximately 200,000 in the absence of MgATP and L-glutamate. Association with the glutamine- and acetylglutamate-dependent carbamoyl phosphate synthetase, also located in the mitochondria, could not be demonstrated. The subunit molecular weight is approximately 46,000. The pH optimum of the biosynthesis reaction is 7.1-7.4. The purified enzyme is stabilized by MgATP and glutamate and by ethylene glycol, and is activated by 5-10% ethylene glycol. The apparent Km values for MgATP, L-glutamate, and ammonia (NH4+-NH3) are 0.7, 11.0, and 0.015 mM, respectively. Mg2+ in excess of that required to complex ATP as MgATP is required for maximal activity; Mn2+ cannot replace Mg2+. The enzyme is activated by low concentrations of chloride, bromide, or iodide; this effect appears to be related to decreases in the apparent Km for glutamate. The enzyme is inhibited by physiological concentrations of urea, but is not significantly affected by physiological concentrations of trimethylamine-N-oxide. Except for activation by halogen anions and the very low apparent Km for ammonia, this elasmobranch glutamine synthetase has properties similar to those reported for mammalian and avian glutamine synthetases. The very low apparent Km for ammonia may be specifically related to the unique role of this glutamine synthetase in mitochondrial assimilation of ammonia for urea synthesis.     

Purification and properties of ornithine carbamoyl transferase from liver of Squalus acanthias

Citrulline synthesis from ammonia by hepatic mitochondria in elasmobranchs involves intermediate formation of glutamine as the result of the presence of high levels of glutamine synthetase and a unique glutamine- and N-acetyl-glutamate-dependent carbamoyl phosphate synthetase, both of which have properties unique to the function of glutamine-dependent synthesis of urea, which is retained in the tissues of elasmobranchs at high concentrations for the purpose of osmoregulation [P.M. Anderson and C.A. Casey (1984) J. Biol. Chem. 259, 456-462; R.A. Shankar and P.M. Anderson (1985) Arch. Biochem. Biophys. 239, 248-259]. The objective of this study was to determine if ornithine carbamoyl transferase, which catalyzes the last step of mitochondrial citrulline synthesis and which has not been previously isolated from any species of fish, also has properties uniquely related to this function. Ornithine carbamoyl transferase was highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme is a trimer with a subunit molecular weight of 38,000 and a native molecular weight of about 114,000. The effect of pH is significantly influenced by ornithine concentration; optimal activity is at pH 7.8 when ornithine is saturating. The apparent Km values for ornithine and carbamoyl phosphate at pH 7.8 are 0.71 and 0.05 mM, respectively. Ornithine displays considerable substrate inhibition above pH 7.8. The activity is not significantly affected by physiological concentrations of the osmolyte urea or trimethylamine-N-oxide or by a number of other metabolites. The results of kinetic studies are consistent with a steady-state ordered addition of substrates (carbamoyl phosphate binding first) and rapid equilibrium random release of products. Except for an unusually low specific activity, the properties of the purified elasmobranch enzyme are similar to the properties of ornithine carbamoyl transferase from mammalian ureotelic and other species and do not appear to be unique to its role in glutamine-dependent synthesis of urea for the purpose of osmoregulation.     

Branchial mitochondria-rich cells in the dogfish Squalus acanthias

In marine teleost fishes, the gill mitochondria-rich cells (MRCs) are responsible for NaCl elimination; however, in elasmobranch fishes, the specialized rectal gland is considered to be the most important site for salt secretion. The role of the gills in elasmobranch ion regulation, although clearly shown to be secondary, is not well characterized. In the present study, we investigated some morphological properties of the branchial MRCs and the localization, and activity of the important ionoregulatory enzyme Na(+)/K(+)-ATPase, under control conditions and following rectal gland removal (1 month) in the spiny dogfish, Squalus acanthias. A clear correlation can be made between MRC numbers and the levels of Na(+)/K(+)-ATPase activity in crude gill homogenates (r(2)=-0.69). Strong Na(+)/K(+)-ATPase immunoreactivity is also clearly associated with the basolateral membrane of these MRCs. In addition, the dogfish were able to maintain ionic balance after rectal gland removal. These results all suggest a possible role of the dogfish gill in salt secretion. MRCs were, however, unresponsive to rectal gland removal in terms of changes in number, fine structure and Na(+)/K(+)-ATPase activity, as might be expected if they were compensating for the loss of salt secretion by the rectal gland. Thus, the specific role that these MRCs play in ion regulation in the dogfish remains to be determined     

Low mass-specific brain Na+/K+-ATPase activity in elasmobranch compared to teleost fishes: implications for the large brain size of elasmobranchs

Elasmobranch fishes have long been noted for having unusually large brains for ectotherms, and therefore may be exceptions to the rule that vertebrates in general devote less than 8% of their resting metabolic rate to the central nervous system. The brain mass of sharks, skates and rays is often several times larger than that of teleost fishes of the same size. Still, the underlying reasons for this have remained unclear. Ion pumping by the Na+/K+-ATPase is the single most energy consuming process in the brain. In this study, Na+/K+-ATPase activity was measured in the brain of four species of elasmobranchs and 11 species of teleosts. While the average brain mass of the elasmobranchs examined was approximately three times that of the teleosts, the mean specific Na+/K+-ATPase activity was only about one-third of that of the teleosts. Thus, the total brain Na+/K+-ATPase activity was similar in elasmobranchs and teleosts. This suggests that the large brain size of elasmobranchs is at least partly related to a low mass-specific rate of brain energy use.     

Carrier-mediated urea transport across the mitochondrial membrane of an elasmobranch (Raja erinacea) and a teleost (Oncorhynchus mykiss) fish

In osmoregulating teleost fish, urea is a minor nitrogen excretory product, whereas in osmoconforming marine elasmobranchs it serves as the major tissue organic solute and is retained at relatively high concentrations ( approximately 400 mmol/l). We tested the hypothesis that urea transport across liver mitochondria is carrier mediated in both teleost and elasmobranch fishes. Intact liver mitochondria in rainbow trout (Oncorhynchus mykiss) demonstrated two components of urea uptake, a linear component at high concentrations and a phloretin-sensitive saturable component [Michaelis constant (K(m)) = 0.58 mmol/l; maximal velocity (V(max)) = 0.12 mumol.h(-1).mg protein(-1)] at lower urea concentrations (<5 mmol/l). Similarly, analysis of urea uptake in mitochondria from the little skate (Raja erinacea) revealed a phloretin-sensitive saturable transport (K(m) = 0.34 mmol/l; V(max) = 0.054 mumol.h(-1).mg protein(-1)) at low urea concentrations (<5 mmol/l). Surprisingly, urea transport in skate, but not trout, was sensitive to a variety of classic ionophores and respiration inhibitors, suggesting cation sensitivity. Hence, urea transport was measured in the reverse direction using submitochondrial particles in skate. Transport kinetics, inhibitor response, and pH sensitivity were very similar in skate submitochondrial particle submitochondrial particles (K(m) = 0.65 mmol/l, V(max) = 0.058 mumol.h(-1).mg protein(-1)) relative to intact mitochondria. We conclude that urea influx and efflux in skate mitochondria is dependent, in part, on a bidirectional proton-sensitive mechanism similar to bacterial urea transporters and reminiscent of their ancestral origins. Rapid equilibration of urea across the mitochondrial membrane may be vital for cell osmoregulation (elasmobranch) or nitrogen waste excretion (teleost).     

The relationships of cartilaginous fishes: an immunological study of serum transferrins of holocephalans and elasmobranchs

Serum transferrins from two holocephalan, five elasmobranch and three teleost species have been compared using quantitative microcomplement fixation. Calculated immunological distances emphasize the close relationship between the holocephalans and elasmobranchs and strongly support the view that they should be considered as part of a natural assemblage which is widely separated from the teleosts.
If the Holocephali and elasmobranchs have been separate since the beginning of the Carboniferous, this implies that transferrin has evolved at a rate approximating to 0-15-0-26 immunological units per million years involving some 9–15% substitution of amino acids. These values are extremely low and indicate that holocephalan and elasmobranch transferrins have evolved some 2–3–7 times more slowly than those known from any other group of vertebrates.     

Freshwater elasmobranchs: a review of their physiology and biochemistry

Only 5% of elasmobranch species live in freshwater (FW) compared to more than 40% of known teleost species. The factors affecting the poor penetration of elasmobranchs into FW environments are currently unknown, however, an important consideration may be the high urea requirement of many proteins in marine elasmobranchs. Urea is an important osmolyte in marine elasmobranchs and must be reduced in dilute environments. There are three identifiable stages in the successful colonization of FW. The euryhaline marine species freely entering and leaving FW represent the initial stage of FW colonization. In this group, there is an apparent inability to eliminate all urea due to protein integrity issues and this results in energy and nitrogen losses that may constrain growth and reproduction. The second stage is represented by those species that live entirely in FW but must also retain some urea. This group also suffers from the same constraints as the first group. These two groups have kidneys and sensory organs that more closely resemble strictly marine forms. The third and final stage is represented by the Potamotrygonid stingrays where the need for urea in FW has been eliminated. Consequently nitrogen and energy losses are reduced and those sections of the kidney needed for urea conservation have been eliminated. The driving force for such modifications is a reduction in urea levels and the concomitant saving of energy needed for urea synthesis. Other physiological adaptations associated with survival in FW include giving birth to live young, the capacity of sperm to be activated in freshwater and modifications of the electrosensory system to function in a low conductivity environment. The need for many anatomical, metabolic and physiological modifications for FW existence may constrain the rapidity and hence the frequency of FW colonization, compared to the situation in the more advanced osmoregulating teleosts. Once optimally adapted to FW, recolonization of sea water by elasmobranchs is problematic due to the loss of urea synthetic capacity and renal structures for urea retention.     

The putative mechanism of Na(+) absorption in euryhaline elasmobranchs exists in the gills of a stenohaline marine elasmobranch, Squalus acanthias

We recently cloned an NHE3 orthologue from the gills of the euryhaline Atlantic stingray (Dasyatis sabina), and generated a stingray NHE3 antibody to unequivocally localize the exchanger to the apical side of epithelial cells that are rich with Na(+)/K(+)-ATPase (A MRC). We also demonstrated an increase in NHE3 expression when stingrays are in fresh water, suggesting that NHE3 is responsible for active Na(+) absorption. However, the vast majority of elasmobranchs are only found in marine environments. In the current study, immunohistochemistry with the stingray NHE3 antibody was used to localize the exchanger in the gills of the stenohaline marine spiny dogfish shark (Squalus acanthias). NHE3 immunoreactivity was confined to the apical side of cells with basolateral Na(+)/K(+)-ATPase and was excluded from cells with high levels of vacuolar H(+)-ATPase. Western blots detected a single protein of 88 kDa in dogfish gills, the same size as NHE3 in stingrays and mammals. These immunological data demonstrate that the putative cell type responsible for active Na(+) absorption in euryhaline elasmobranchs is also present in stenohaline marine elasmobranchs, and suggest that the inability of most elasmobranchs to survive in fresh water is not due to a lack of the gill ion transporters for Na(+) absorption.     

The control of drinking in elasmobranch fish with special reference to the renin-angiotensin system

In summary, it is evident that teleost and elasmobranch fish respond to extra-cellular dehydration by increasing drinking rate mediated by an increase in circulating levels of ANG II. However, although the primary stimulus for drinking may be the same, clearly the mechanisms involved in regulating ion and water balance are entirely different. In order to maintain ion and water balance in the face of cellular and extra-cellular dehydration, the integration and hormonal control of renal and extra-renal function in elasmobranchs has developed in a very different manner to that described for teleost fish.     

Urea and chloride sensitivities of coelacanth hemoglobin

Synopsis At pH 6.96–6.98, 20°C and in the absence of inorganic ions, the O₂ affinity of thawed Latimeria chalumnae hemoglobin was 1.53–1.86 mmHg; cooperativity was 1.00–1.13. These values are essentially the same as those in the literature for samples that had never been frozen. There was no clear effect of either urea (up to> 3M) or KCl (up to> 1M) on O₂ binding. Thus the hemoglobins of the coelacanth, as well as those of most of the elasmobranchs examined, are insensitive to urea, a major intracellular osmolyte in these groups and a denaturing agent in higher vertebrates. However, the absence of comparable information on more primitive hemoglobins and also on teleost hemoglobins precludes a clear evolutionary interpretation of the origin of urea sensitivity of the hemoglobins in higher vertebrates.     

Characteristics of the conditioned motor reactions of unrestrained elasmobranches and teleosts

In elasmobranch (Scyllium canicula, Galleus canis) and teleost (Migull capitocum) fishes it is possible to form motor food-searching conditioned reflexes to discrimination of light and darkness. Some differences were revealed in ecological and conditioned motor behavioural activities in higher and lower sharks. Elasmobranch and teleost fishes exhibit significant differences in ecological, feeding and conditioned reflex behaviour. Nervous activity in elasmobranchs is characterized by lower and primitive organization as compared to that in teleosts.     

Antagonistic effect of urea on oxygenation-linked binding of ATP in an elasmobranch hemoglobin

The O2 affinity of "stripped" (cofactor-free) hemoglobin (Hb) of the elasmobranch, Squalus acanthias is decreased by ATP, the main erythrocytic phosphate cofactor but increased by urea at physiological concentration. When both compounds are present, as in life, urea decreases the ATP sensitivity, indicating that previous Hb oxygenation studies in the absence of urea overestimate the modulator role of phosphate cofactors in sharks. Whereas ATP decreases the O2 association equilibrium constant of the deoxygenated pigment, urea raises those of both the deoxy and the oxygenated states. Possible mechanisms for the urea-protein interactions i.e. binding at carboxy-termini or carbamylation of amino-termini of the protein chains, are discussed.     

Urea based osmoregulation and endocrine control in elasmobranch fish with special reference to euryhalinity

Since the landmark contributions of Homer Smith and co-workers in the 1930s there has been a considerable advance in our knowledge regarding the osmoregulatory strategy of elasmobranch fish. Smith recognised that urea was retained in the body fluids as part of the ‘osmoregulatory ballast’ of elasmobranch fish so that body fluid osmolality is raised to a level that is iso- or slightly hyper-osmotic to that of the surrounding medium. From studies at that time he also postulated that many marine dwelling elasmobranchs were not capable of adaptation to dilute environments. However, more recent investigations have demonstrated that, at least in some species, this may not be the case. Gradual acclimation of marine dwelling elasmobranchs to varying environmental salinities under laboratory conditions has demonstrated that these fish do have the capacity to acclimate to changes in salinity through independent regulation of Na⁺, Cl⁻ and urea levels. This suggests that many of the presumed stenohaline marine elasmobranchs could in fact be described as partially euryhaline. The contributions of Thomas Thorson in the 1970s demonstrated the osmoregulatory strategy of a fully euryhaline elasmobranch, the bull shark, Carcharhinus leucas, and more recent investigations have examined the mechanisms behind this strategy in the euryhaline elasmobranch, Dasyatis sabina. Both partially euryhaline and fully euryhaline species utilise the same physiological processes to control urea, Na⁺ and Cl⁻ levels within the body fluids. The role of the gills, kidney, liver, rectal gland and drinking process is discussed in relation to the endocrine control of urea, Na⁺ and Cl⁻ levels as elasmobranchs acclimate to different environmental salinities.     

Reply to Hussey et al.: The requirement for accurate diet-tissue discrimination factors for interpreting stable isotopes in sharks

Carbon and nitrogen stable isotope analyses have improved our understanding of food webs and movement patterns of aquatic organisms. These techniques have recently been applied to diet studies of elasmobranch fishes, but isotope turnover rates and isotope diet–tissue discrimination are still poorly understood for this group. We performed a diet switch experiment on captive sandbar sharks (Carcharhinus plumbeus) as a model shark species to determine tissue turnover rates for liver, whole blood, and white muscle. In a second experiment, we subjected captive coastal skates (Leucoraja spp.) to serial salinity reductions to measure possible impacts of tissue urea content on nitrogen stable isotope values. We extracted urea from spiny dogfish (Squalus acanthias) white muscle to test for effects on nitrogen stable isotopes. Isotope turnover was slow for shark tissues and similar to previously published estimates for stingrays and teleost fishes with low growth rates. Muscle isotope data would likely fail to capture seasonal migrations or diet switches in sharks, while liver and whole blood would more closely reflect shorter term movement or shifts in diet. Nitrogen stable isotope values of skate blood and skate and dogfish white muscle were not affected by tissue urea content, suggesting that available diet–tissue discrimination estimates for teleost fishes with similar physiologies would provide accurate estimates for elasmobranchs.     

Active urea transport and an unusual basolateral membrane composition in the gills of a marine elasmobranch

In elasmobranch fishes, urea occurs at high concentrations (350-600 mM) in the body fluids and tissues, where it plays an important role in osmoregulation. Retention of urea by the gill against this huge blood-to-water diffusion gradient requires specialized adaptations to the epithelial cell membranes. Experiments were performed to determine the mechanisms and structural features that facilitate urea retention by the gill of the spiny dogfish Squalus acanthias. Analysis of urea uptake by gill basolateral membrane vesicles revealed the presence of a phloretin-sensitive (half inhibition 0.09 mM), sodium-coupled, secondary active urea transporter (Michaelis constant = 10.1 mM, maximal velocity = 0.34 micromol. h(-1). mg protein(-1)). We propose that this system actively transports urea out of the gill epithelial cells back into the blood against the urea concentration gradient. Lipid analyses of the basolateral membrane revealed high levels of cholesterol contributing to the highest reported cholesterol-to-phospholipid molar ratio (3.68). This unique combination of active urea transport and modification of the phospholipid bilayer membrane is responsible for decreasing the gill permeability to urea and facilitating urea retention by the gill of Squalus acanthias.     

Effect of phosphorylation on the actin-activated atpase activity of myosin

The purpose of this study was to test the hypothesis that the phosphorylation of myosin is solely responsible for the activation of the Mg²⁺-ATPase activity of gizzard actomyosin. Using a washed natural actomyosin and a reconstituted actomyosin it was shown that phosphorylation alone caused only a slight activation of ATPase activity. Full activity was obtained only when proteins in addition to the myosin light chain kinase were added. It is evident from these results that: 1) there is no simple relationship between the extent of myosin phosphorylation and the specific Mg²⁺-ATPase activity of actomyosin and 2) in order for full activation by actin of the Mg²⁺-ATPase activity of phosphorylated myosin additional factors are required.     

Effects of urea and guanidine hydrochloride on the sliding movement of actin filaments with ATP hydrolysis by myosin molecules

To evaluate the role of the hydration layer on the protein surface of actomyosin, we compared the effects of urea and guanidine-HCl on the sliding velocities and ATPase activities of the actin-heavy meromyosin (HMM) system. Both chemicals denature proteins, but only urea perturbs the hydration layer. Both the sliding velocity of actin filaments and actin-activated ATPase activity decreased with increasing urea concentrations. The sliding movement was completely inhibited at 1.0 M urea, while actin filaments were bound to HMM molecules fixed on the glass surface. Guanidine-HCl (0-0.05 M) drastically decreased both the sliding velocity and ATPase activation of acto-HMM complexes. Under this condition, actin filaments almost detached from HMM molecules. In contrast, the ATPase activity of HMM without actin filaments was almost independent of urea concentrations <1.0 M and guanidine-HCl concentrations <0.05 M. An increase in urea concentrations up to 2.0 M partly induced changes in the ternary structure of HMM molecules, while the actin filaments were stable in this concentration range. Hydration changes around such actomyosin complexes may alter both the stability of part of the myosin molecules, and the affinity for force transmission between actin filaments and myosin heads.