Paul H. Yancey

Unusual organic osmolytes in deep-sea animals: adaptations to hydrostatic pressure and other perturbants.

Shallow-living marine invertebrates use free amino acids as cellular osmolytes, while most teleosts use almost no organic osmolytes. Recently we found unusual osmolyte compositions in deep-sea animals. Trimethylamine N-oxide (TMAO) increases with depth in muscles of some teleosts, skates, and crustaceans (up to 300 mmol/kg at 2900 m). Other deep-sea animals had high levels of (1). scyllo-inositol in echinoderms, gastropods, and polychaetes, (2). that polyol plus beta-alanine and betaine in octopods, (3). hypotaurine, N-methyltaurine, and unidentified methylamines in vestimentiferans from hydrothermal vents and cold seeps, and (4). a depth-correlated serine-phosphate osmolyte in vesicomyid clams from trench seeps. We hypothesize that some of these solutes counteract effects of hydrostatic pressure. With lactate dehydrogenase, actin, and pyruvate kinase, 250 mM TMAO (but not glycine) protected both ligand binding and protein stability against pressure. To test TMAO in living cells, we grew yeast under pressure. After 1 h at 71 MPa, 3.5 h at 71 MPa, and 17 h at 30 MPa, 150 mM TMAO generally doubled the number of cells that formed colonies. Sulfur-based osmolytes which are not correlated with depth, such as hypotaurine and thiotaurine, are probably involved in sulfide metabolism and detoxification. Thus deep-sea osmolytes may have at least two other roles beyond acting as simple compatible osmotica.

High Contents of Trimethylamine Oxide Correlating With Depth in Deep-Sea Teleost Fishes, Skates, and Decapod Crustaceans

In muscles of shallow-living marine animals, the osmolyte trimethylamine N-oxide (TMAO) is reportedly found (in millimoles of TMAO per kilogram of tissue wet weight) at 30-90 in shrimp, 5-50 in crabs, 61-181 in skates, and 10-70 in most teleost fish. Recently our laboratory reported higher levels (83-211 mmol/kg), correlating with habitat depth, in deep-sea gadiform teleosts. We now report the same trend in muscles of other animals, collected off the coast of Oregon from bathyal (1800-2000 m) and abyssal plain (2850 m) sites. TMAO contents (mmol/kg +/- SD) were as follows: zoarcid teleosts, 103 +/- 9 (bathyal) and 197 +/- 2 (abyssal); scorpaenid teleosts, 32 +/- 0 (shallow) and 141 +/- 16 (bathyal); rajid skates, 215 +/- 13 (bathyal) and 244 +/- 23 (abyssal); caridean shrimp, 76 +/- 16 (shallow), 203 +/- 35 (bathyal), and 299 +/- 28 (abyssal); Chionoecetes crabs, 22 +/- 2 (shallow) and 164 +/- 15 (bathyal). Deep squid, clams, and anemones also had higher contents than shallow species. Osmoconformers showed compensation between TMAO and other osmolytes. Urea contents (typically 300 mmol/kg in shallow elasmobranchs) in skates were 214 +/- 5 (bathyal) and 136 +/- 9 (abyssal). Glycine contents in shrimp were 188 +/- 17 (shallow) and 52 +/- 20 (abyssal). High TMAO contents may reflect diet, reduce osmoregulatory costs, increase buoyancy, or counteract destabilization of proteins by pressure.

Trimethylamine oxide counteracts effects of hydrostatic pressure on proteins of deep‐sea teleosts

In shallow marine teleost fishes, the osmolyte trimethylamine oxide (TMAO) is typically found at <70 mmol/kg wet weight. Recently we found deep-sea teleosts have up to 288 mmol/kg, increasing in the order shallow < bathyal < abyssal. We hypothesized that this protein stabilizer counteracts inhibition of proteins by hydrostatic pressure, and showed that, for lactate dehydrogenases (LDH), 250 mM TMAO fully offset an increase in NADH K(m) at physiological pressure, and partly reversed pressure-enhanced losses of activity at supranormal pressures. In this study, we examined other effects of pressure and TMAO on proteins of teleosts that live from 2000-5000 m (200-500 atmospheres [atm]). First, for LDH from a grenadier (Coryphaenoides leptolepis) at 500 atm for 8 hr, there was a significant 15% loss in activity (P < 0.05 relative to 1 atm control) that was reduced with 250 mM TMAO to an insignificant loss. Second, for pyruvate kinase from a morid cod (Antimora microlepis) at 200 atm, there was 73% increase in ADP K(m) without TMAO (P < 0.01 relative to K(m) at 1 atm) but only a 29% increase with 300 mM TMAO. Third, for G-actin from a grenadier (C. armatus) at 500 atm for 16 hr, there was a significant reduction of F-actin polymerization (P < 0.01 compared to polymerization at 1 atm) that was fully counteracted by 250 mM TMAO, but was unchanged in 250 mM glycine. These findings support the hypothesis. J. Exp. Zool. 289:172-176, 2001.

Urea-requiring lactate dehydrogenases of marine elasmobranch fishes

SummaryThe kinetic properties — apparentKm of pyruvate, pyruvate inhibition pattern, and maximal velocity — of M4 (skeletal muscle) lactate dehydrogenases of marine elasmobranch fishes resemble those of the homologous lactate dehydrogenases of non-elasmobranchs only when physiological concentrations of urea (approximately 400 mM) are present in the assay medium. Urea increases the apparentKm of pyruvate to values typical of other vertebrates (Fig. 2), and reduces pyruvate inhibition to levels seen with other M4-lactate dehydrogenases (Fig. 3). Urea reduces the activation enthalpy of the reaction, and increasesVmax at physiological temperatures (Fig. 4).The M4-lactate dehydrogenase of the freshwater elasmobranch,Potamotrygon sp., resembles a teleost lactate dehydrogenase, i.e., although it is sensitive to urea, it does not require the presence of urea for the establishment of optimal kinetic properties.

Developmental changes in organic osmolytes in prenatal and postnatal rat tissues.

At high osmotic pressures, mammalian kidney medulla, heart, lens, and brain utilize organic osmolytes to regulate cell volume. However the types and proportions of these solutes vary among tissues in patterns and for non-osmotic roles not fully elucidated. To clarify these, we analyzed osmolyte-type solute contents in rat tissues at 7 and 2 days prenatal and at 0, 7, 14, 21 (weaning), 35 (juvenile) and 77 (adult) days postnatal. Placentas were dominated by betaine, taurine, and creatine, which decreased between the prenatal times. Fetuses were dominated by glutamate and taurine, which increased between the times. In cerebrum, hindbrain and diencephalon, taurine dominated at early stages, but dropped after postnatal day 7, while myo-inositol, glutamine, creatine and glutamate increased after birth, with the latter two dominating in adults. In olfactory bulb, taurine content declined gradually with age and was equal to glutamate in adults. In all brain regions, glycerophosphorylcholine (GPC) reached a peak in juveniles. In postnatal renal medulla, urea, sodium, GPC, betaine, and taurine increased sharply at day 21. Thereafter, most increased, but taurine decreased. In heart, taurine dominated, and increased with age along with creatine and glutamine, while glutamate decreased after postnatal day 7. In lens, taurine dominated and declined in adults. These patterns are discussed in light of hypotheses on non-osmotic and pathological roles of these solutes.

Elevated levels of trimethylamine oxide in muscles of deep‐sea gadiform teleosts: A high‐pressure adaptation?

Trimethylamine oxide (TMAO) as an osmolyte typically occurs at 20–70 mM in shallow-water marine teleost fishes. However, it has not been previously examined in deep-living species. We collected species from two families of benthic gadiform teleosts by otter trawl from the continental slope (1,800–2,000 m) and abyssal plain (2,850 m) off the Oregon coast. Muscle and plasma samples were analyzed for TMAO with a picric acid method. Muscle contents (below in millimoles/kilogram of wet weight) were found to be higher than previously reported for teleosts. Results from Macrouridae from 1,800–2,200 m were as follows: Albatrossia pectoralis, 83 ± 10; Coryphaenoides cinerus, 121 ± 11. Results from Macrouridae from 2,850 m were significantly higher: C. leptolepis, 158 ± 20; C. fillifer, 177 ± 8; C. armatus, 173 ± 5. Caught at both depths, Antimora microlepis (Moridae) had 211 ± 14. Plasmas had low TMAO (2–15 mM) and high Na+concentrations (227–273 mM), except A. microlepis plasma, which had the lowest Na+ (219 ± 30 mM) and highest TMAO (159 ± 46 mM). Osmotic pressures of fresh plasma (423–557 mosm) correlated highly with muscle TMAO and with plasma TMAO plus Na+ levels. These higher osmolalities may reduce osmoregulatory costs. However, as a methylamine known to stabilize protein, TMAO may counteract destabilizing effects of hydrostatic pressure on cellular proteins. With purified C. leptolepis muscle lactate dehydrogenase, 250 mM TMAO fully offset a 30% increase in NADH Km induced by 300 atm. J. Exp. Zool. 279:386–391, 1997.

Osmotic effectors in kidneys of xeric and mesic rodents: corticomedullary distributions and changes with water availability.

SummaryUrea, sodium, the methylamines glycine betaine and glycerophosphorylcholine (GPC), and the polyols sorbitol and myo-inositol are reported to be the major osmolytes in kidneys of laboratory mammals. These were measured (millimoles per kilogram wet weight) in kidney regions and urines of three species of wild rodents with different dehydration tolerances: the pocket mousePerognathus parvus (xeric), voleMicrotus montanus (mesic), and deer mousePeromyscus m. gambeli (intermediate). In animals kept without water for 4–6 days, sodium, urea, betaine and GPC+choline were found in gradients increasing from cortex to outer to inner medulla in all species, withPerognathus having the highest levels. Sorbitol was high in the inner medulla but low in the cortex and outer medulla; inositol was highest in the outer medulla. Totals of methylamines and methylamines plus polyols in the medulla showed high linear correlations (positive) with urea and with sodium values.Whole medullae were analyzed at several time points inMicrotus andPeromyscus subject to water diuresis followed by antidiuresis. In 102 h diuresis inMicrotus, all osmolytes decreased except inositol; however, only urea, sodium and sorbitol reached new steady states within 24 h. Urea returned to initial values in 18 h antidiuresis, while other osmolytes required up to 90 h. InPeromyscus, all osmolytes except the polyols declined in diuresis (max. 78 h test period). During antidiuresis, urea and GPC+choline rose to initial values in 18 h, with sodium and betaine requiring more time. In plots of both species combined, total methylamines+polyols correlated linearly (positive) with sodium, and GPC+choline with urea.Estimates of tissue concentrations suggest that total methylamines+polyols can account for intracellular osmotic balance in all species in antidiuresis and that sufficient concentrations of methylamines may be present to counteract perturbing effects of urea on proteins.

Compatible and counteracting solutes: protecting cells from the Dead Sea to the deep sea.

Cells of many organisms accumulate certain small organic molecules -called compatible and counteracting solutes, compensatory solutes, or chemical chaperones – in response to certain physical stresses. These solutes include certain carbohydrates, amino acids, methylamine and methylsulphonium zwitterions, and urea. In osmotic dehydrating stress, these solutes serve as cellular osmolytes. Unlike common salt ions and urea (which inhibit proteins), some organic osmolytes are compatible; i.e., they do not perturb macromolecules such as proteins. In addition, some may protect cells through metabolic processes such as antioxidation reactions and sulphide detoxification. Other osmolytes, and identical or similar solutes accumulated in anhydrobiotic, heat and pressure stresses, are termed counteracting solutes or chemical chaperones because they stabilise proteins and counteract protein-destabilising factors such as urea, temperature, salt, and hydrostatic pressure. Stabilisation of proteins, not necessarily beneficial in the absence of a perturbant, may result indirectly from effects on water structure. Osmotic shrinkage of cells activates genes for chaperone proteins and osmolytes by mechanisms still being elucidated. These solutes have applications in agriculture, medicine and biotechnology.

Marine fish may be biochemically constrained from inhabiting the deepest ocean depths

Significance Fish appear to be absent from the oceans greatest depths, the trenches from 8,400–11,000 m. The reason is unknown, but hydrostatic pressure is suspected. We propose that the answer is the need for high levels of trimethylamine oxide (TMAO, common in many marine animals), a potent stabilizer capable of counteracting the destabilization of proteins by pressure. TMAO is known to increase with depth in bony fishes (teleosts) down to 4,900 m. By capturing the worlds second-deepest known fish, the hadal snailfish Notoliparis kermadecensis from 7,000 m, we find that they have the highest recorded TMAO contents, which, moreover, yield an extrapolated maximum for fish at about 8,200 m. This is previously unidentified evidence that biochemistry may constrain depth for a large taxonomic group. No fish have been found in the deepest 25% of the ocean (8,400–11,000 m). This apparent absence has been attributed to hydrostatic pressure, although direct evidence is wanting because of the lack of deepest-living species to study. The common osmolyte trimethylamine N-oxide (TMAO) stabilizes proteins against pressure and increases with depth, going from 40 to 261 mmol/kg in teleost fishes from 0 to 4,850 m. TMAO accumulation with depth results in increasing internal osmolality (typically 350 mOsmol/kg in shallow species compared with seawaters 1,100 mOsmol/kg). Preliminary extrapolation of osmolalities of predicted isosmotic state at 8,000–8,500 m may indicate a possible physiological limit, as greater depths would require reversal of osmotic gradients and, thus, osmoregulatory systems. We tested this prediction by capturing five of the second-deepest known fish, the hadal snailfish (Notoliparis kermadecensis; Liparidae), from 7,000 m in the Kermadec Trench. We found their muscles to have a TMAO content of 386 ± 18 mmol/kg and osmolality of 991 ± 22 mOsmol/kg. These data fit previous extrapolations and, combined with new osmolalities from bathyal and abyssal fishes, predict isosmotic state at 8,200 m. This is previously unidentified evidence that biochemistry could constrain the depth of a large, complex taxonomic group.

Correlation of Trimethylamine Oxide and Habitat Depth within and among Species of Teleost Fish: An Analysis of Causation

Most shallow‐water teleosts have moderate levels of trimethylamine N‐oxide (TMAO; ∼50 mmol/kg wet mass), a common osmolyte in many other marine animals. Recently, muscle TMAO contents were found to increase linearly with depth in six families. In one hypothesis, this may be an adaptation to counteract the deleterious effects of pressure on protein function, which TMAO does in vitro. In another hypothesis, TMAO may be accumulated as a by‐product of acylglycerol (AG) production, increasing with depth because of elevated lipid metabolisms known to occur in some deep‐sea animals. Here we analyze muscle TMAO contents and total body AG (mainly triacyglycerol [TAG]) levels in 15 species of teleosts from a greater variety of depths than sampled previously, including eight individual species caught at two or more depths. Including data of previous studies (total of 17 species, nine families), there is an apparent sigmoidal increase in TMAO contents between 0‐ and 1.4‐km depths, from about 40 to 150 mmol/kg. From 1.4 to 4.8 km, the increase appears to be linear ( \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \renewcommand\encodingdefault{OT2} \normalfont \selectfont} \DeclareTextFontCommand{\textcyr}{\cyr} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \landscape