4 - Organic Osmolytes in Elasmobranchs

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  • 1.

    Introduction

  • 2.

    Osmoconformers Versus Osmoregulators

    • 2.1.

      Osmoconforming: Elasmobranchs in the Oceans

    • 2.2.

      Osmoregulating: Elasmobranchs at Low Salinities

  • 3.

    Properties of Organic Osmolytes

    • 3.1.

      Inorganic Ions Versus Compatible Solutes

    • 3.2.

      Urea Versus Methylamines: Counteracting Solutes

    • 3.3.

      Salts Versus Methylamines: Counteracting Solutes

    • 3.4.

      TMAO Versus Pressure: A Piezolyte in the Deep Sea?

    • 3.5.

      Physicochemical Mechanisms of (De)stabilization

    • 3.6.

      Urea and Methylamines: Buoyancy

    • 3.7.

      Other Cytoprotective/Compensatory Properties

    • 3.8.

      Implications for Food Industry

  • 4.

    Metabolism and Regulation

    • 4.1.

      Osmolyte Synthesis Versus Dietary Intake

    • 4.2.

      Retention of Osmolytes

    • 4.3.

      Development

    • 4.4.

      Cell Volume Regulation

    • 4.5.

      Hormonal Regulation

  • 5.

    Evolutionary Considerations

  • 6.

    Knowledge Gaps and Future Directions

Marine elasmobranchs are hypoionic osmoconformers. Their extra- and intracellular fluids accumulate organic osmolytes for osmotic balance, mainly urea and methylamines such as trimethylamine N-oxide (TMAO), higher intracellularly at about 2:1 in shallow species, even in embryos and starvation. The relatively few euryhaline species reduce osmolytes only partially, and become hyperosmotic regulators, while permanent freshwater species have almost no organic osmolytes. Urea binds to and unfolds proteins while methylamines promote folding, thermodynamically counteracting each other at 2:1. Both also provide buoyancy. Deep-sea elasmobranchs increase TMAO and reduce urea, possibly because TMAO also counteracts protein-destabilizing effects of hydrostatic pressure. Recent studies show TMAO coordinates water molecules in a complex excluded from protein backbones (“osmophobicity”). This entropically favors folded protein conformations, and conteracts urea and pressure unfolding effects. Osmolytes are made in the liver or obtained from diets and retained by adaptations of gill, kidney, intestine, rectal gland. Regulatory mechanisms and evolutionary history are incompletely understood.

Introduction

Osmotic balance – especially for maintenance of cell volume – is a fundamental challenge of homeostasis for all organisms. Excessive swelling or shrinking of cells, due to osmosis across semipermeable plasma membranes, changes cellular concentrations (which in turn alters reaction rates, ion interactions with macromolecules, etc.), and may mechanically damage membranes. Cell shrinkage occurs from evaporation or hyperosmotic solutions (external solutions with solute concentrations higher than the cell), while swelling occurs in hypo-osmotic solutions such as freshwater. The potential for swelling damage is worse in animals than in organisms with rigid cell walls that prevent osmotic swelling by turgor pressure.

First, some definitions are in order. The focus of this chapter is osmolytes: small molecules whose concentrations are used to maintain cell volume by balancing intra- and extracellular solute concentrations and are typically regulated in response to osmotic disturbances. Familiar examples of osmolytes are salt ions – Na+ plus Cl – whose concentrations in mammalian plasma are tightly regulated in part to protect cells from swelling and shrinking.

How do osmolytes work? Recall the four colligative properties of solutions – raised boiling point, depressed freezing point, lowered vapor pressure, and osmotic pressure – that depend only on the number of dissolved particles in solution and not on their structure, size, or mass. Most relevant here is osmotic pressure, defined as the hydraulic pressure (in units of atmosphere, torr, mm Hg, or pascal) needed to prevent osmosis (water movement across a semipermeable membrane) from an area of low to an area of high solute concentration. The osmotic effect of a dissolved particle is its osmotic coefficient, exactly 1.0 for an ideal solute. Importantly, a dissociating compound splits into multiple particles in solution. Thus, while glucose has a coefficient of 1, NaCl (Na+ plus Cl in solution) would be expected to have a coefficient of 2 and NaH2PO4 a coefficient of 4 (Na+, H+, H+ and PO43− in solution) (however, these salts are not ideal; as explained in the following).

While plant physiologists often measure turgor osmotic pressures, animal biologists are rarely able to measure pressures across membranes, so characterizations of osmotic (im)balances are typically expressed in terms of two related properties – osmolality or osmolarity, defined as follows.

  • Osmolality is directly proportional to osmotic pressure and thus other colligative properties, and to molality – moles of solute per kilogram of solvent (water). Specifically, 1 millimolal (millimoles/kg water) yields 1 milliosmole/kg water, abbreviated mOsm/kg. Osmolality does not change with temperature or hydrostatic pressure and is measurable by laboratory osmometers (which determine freezing point or vapor pressure and calculate osmolality by colligative equivalence).

  • Osmolarity is proportional to molarity (M) – moles of solute per liter solution (abbreviated Osm, or mOsm for millimolar). Biologists make chemical solutions using molarity, because that is the relevant factor for chemical reactions; thus, many use mOsm rather than mOsm/kg because of familiar units and because one can quickly estimate osmolarity from molarity. For example, a physiologist finding shark plasma with 400 mM urea estimates the osmolarity contribution at 400 mOsm. However, osmolarity is problematic because: (i) unlike osmolality, it is not a colligative property and cannot be measured; (ii) it changes with temperature and pressure; (iii) the amount of solute dissolved changes the volume of water (for most solutes, a 1 M solution has a higher solute concentration than a 1 molal solution); (iv) not all solutes behave ideally, so estimates of mOsm from M values can be significantly off (Robertson, 1989). A biologically relevant example is NaCl: if ideal, its osmotic coefficient would be 2.0, but in reality it is not fully dissociated in solution, so its coefficient is 1.86 at 25°C at mammalian concentrations (Hamer and Wu, 1972). Thus 150 mM NaCl gives 279 mOsm, not 300 mOsm. Even using osmotic coefficients is not sufficient, as they change in complex solute mixtures. Finally, nondissociating organic osmolytes can also have nonideal coefficients (Section 3).

Thus osmometry that empirically yields osmolality is always preferred over osmolarity. These two terms are often confused and indeed, osmolality is often erroneously called osmolarity in many publications (Erstad, 2003). Therefore, osmolality in mOsm/kg will be used for specific data in this chapter; however, because biologists are used to thinking in molarity, mOsm will be used when discussing general patterns. Molarity will also be used when discussing concentrations relevant to solution interactions.

Before turning to elasmobranchs, it is useful to remember three key osmotic benchmarks – 0 mOsm for pure water, 300 mOsm for basic cell solutes, and 1000 mOsm for seawater. As described in Table 4.1, these are the approximate values for the most common internal and habitat osmolarities.

Section snippets

Osmoconformers Versus Osmoregulators

In marine animals, two broad strategies have evolved to deal with the osmotic challenges of a potentially dehydrating environment: (i) osmoconformation – having an internal osmolality similar to the environment; and (ii) osmoregulation – maintaining an internal osmolality significantly different than the environment. The term “osmoregulation” is often used broadly (covering all osmotic systems), but here it will be used in this stricter definition, akin to thermoconformation versus

Properties of Organic Osmolytes

One of the first questions that arose after the discoveries of organic osmolytes is this: why do cells use organic solutes, which may cost energy and/or tie up compounds that have other uses, instead of “free” inorganic ions (especially NaCl) to raise cellular osmolality when needed?

Metabolism and Regulation

Clearly, large amounts of organic osmolytes are required for elasmobranch osmotic balance. Metabolism and regulation of elasmobranch osmolytes have been thoroughly reviewed recently (Hammerschlag, 2006, Trischitta et al., 2012, Gelsleichter and Evans, 2012) including in this book series (Ballantyne and Fraser, 2013), so only a brief version will be given here. (See also Chapters 5 and 7 for more information).

Evolutionary Considerations

Possible scenarios for the evolution of fish osmoregulation/osmoconformation have been presented many times (reviewed by Schultz and McCormick, 2013). Primitive kidney features suggest that protovertebrates evolved in low salinities and/or as euryhaline (Griffith, 1987, Ditrich, 2007). For teleosts, strong physiological, fossil, and (most recently) phylogenetic/molecular evidence indicates that ancestors evolved in freshwater or estuaries (Vega and Wiens, 2012). For stability in varying

Knowledge Gaps and Future Directions

Many questions remain unanswered regarding elasmobranch organic osmolytes, including the following:

  • The evolutionary origins of elasmobranch osmotic physiology

  • Genes, RNAs (including microRNAs), and proteins (membrane channels, synthetases, etc.) for regulation of cellular organic osmolytes: mechanisms and regulation

  • Hormonal and intracellular regulation of plasma organic osmolytes and synthesis

  • Detailed mechanisms of osmolyte properties including co-evolution of protein intrinsic structures with

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