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Osmoregulation: Water and Solute Balance. OUTLINE:. (1) Background: Marine vs Freshwater vs Terrestrial Habitats (2) Osmotic Pressure vs Ionic Concentration (3) How Ionic Gradients and Osmotic constancy are maintained (4) Ion Uptake Mechanisms. The concept of a “Regulator”.

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Osmoregulation: Water and Solute Balance

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Osmoregulation water and solute balance

  • Osmoregulation:

  • Water and Solute Balance


Osmoregulation water and solute balance

OUTLINE:

(1) Background: Marine vs Freshwater vs Terrestrial Habitats

(2) Osmotic Pressurevs Ionic Concentration

(3) HowIonic GradientsandOsmotic constancyare maintained

(4) Ion Uptake Mechanisms


The concept of a regulator

The concept of a “Regulator”


The concept of a regulator1

The concept of a “Regulator”

  • Maintain constancy (homeostasis) in the face of environmental change

  • Could regulate in response to changes in temperature, ionic concentration, pH, oxygen concentration, etc…


Osmoregulatory capacity varies among species

Osmoregulatory capacity varies among species

The degree to which organisms “regulate” varies. Regulation requires energy and the appropriate physiological systems (organs, enzymes, etc)


Osmoregulation water and solute balance

Life evolved in the Sea


Osmoregulation water and solute balance

The invasion of freshwater from marine habitats, and the invasion of land from water constitute among the most dramatic physiological challenges during the history of life on earth

Of the 32+ phyla, only 16 phyla invaded fresh water,

And only 7 phyla have groups that invaded land

  • Platyhelminthes (flat worms)

  • Nemertea (round worms)

  • Annelids (segmented worms)

  • Mollusca (snails)

  • Onychophora

  • Arthropods(insects, spiders, etc)

  • Chordata (vertebrates)


Habitat invasions

Habitat Invasions

SeaFresh waterSoilLand

ProtistaXXX

PoriferaXX

CnideriaXX

CtenophoraX

PlatyhelminthesXXXX

NemerteaXXX

RotiferaXXX

GastrotrichaXX

KinorhynchaX

NematodaXXX

NematomorphaXX

EntoproctaXX

AnnelidaXXXX

MolluscaXXXX

PhoronidaX


Habitat invasions1

Habitat Invasions

SeaFresh waterSoilLand

BryozoaXX

BrachiopodaX

SipunculidaX

EchiuroidaX

PriapulidaX

TardigradaXXX

OnychophoraXX

ArthropodaXXXX

EchinodermataX

ChaetognathaX

PogonophoraX

HemichordataX

ChordataXXXX


Osmoregulation water and solute balance

Fresh Water (vs Marine)

• Lack of ions

• Greater fluctuations in Temperature, Ions, pH

• Life in fresh water is energetically more expensive

Ionic Composition (g/liter)

MarineFresh Water

Na+10.810.0063

Mg++ 1.300.0041

Ca++ 0.41 0.0150

K+ 0.390.0023

Cl-19.440.0078

SO4-2 2.710.0112

CO3-2 0.140.0584


Osmoregulation water and solute balance

OUTLINE:

(1) Background: Marine vs Freshwater vs Terrestrial Habitats

(2) Osmotic Pressure vs Ionic Concentration

(3) How Ionic Gradients and Osmotic constancy are maintained

(4) Ion Uptake Mechanisms


Challenges

Challenges:

  • Osmotic concentration

  • Ionic concentration


Osmoregulation

Osmoregulation

  • The regulation of water and ions poses among the greatest challenges for surviving in different habitats.

  • Marine habitats pose the least challenge, while terrestrial habitats pose the most. In terrestrial habitats must seek both water and ions (food).

  • In Freshwater habitats, ions are limiting while water is not.


Water

WATER

  • Universal Solvent

  • Polar solution in which ions (but not nonpolar molecules) will dissolve

  • Used for transport (blood, etc)

    Animals are 60-80% water

    75% of the water is intracellular

    20% is extracellular (5-10% vascular)

    All the fluids contain solutes


Why do we need ions as free solutes

Why do we need ions as free solutes?

  • Need to maintain Ionic gradients:

  • Produce of Electrical Signals

  • Enables Electron Transport Chain

  • (production of energy)

  • Used for active transport into cell

  • Na+K+ pump (Na,K-ATPase) 25% of total energy expenditure


Why na and k

Why Na+ and K+?

  • Na+ is the most abundant ion in the sea

  • Intracellular K+: K+ is small, dissolves more readily

  • Stabilizes proteins more than Na+


How does ionic composition differ in and out of the cell

How does ionic composition differ in and out of the cell?


Differences between intra and extra cellular fluids

Differences between intra and extra cellular fluids

  • Very different ionic composition

  • (Hi K+ in, Hi Na+ out)

  • Lower inorganic ionic concentration inside

    (negative potential)

  • Osmolytes to compensate for osmotic difference inside cell


Osmoregulation water and solute balance

Extracellular Fluids

Na+

The Cell

HCO3-

K+

Organic

Anions

K+

Mg++

Cl-

Cl-

Mg++

Na+

Ca++

Ca++


Osmoregulation water and solute balance

Extracellular Fluids

Electrochemical

Chemical Gradient

Negative

Potential Inside

Na+

K+

Organic

Anions

K+

Mg++

Mg++

Cl-

Ca++

Cl-

Ca++

Na+

HCO3-


Challenges1

Challenges:

  • Osmotic concentration

  • Ionic concentration


Osmotic concentration

Osmotic Concentration

  • Balance of number of solutes

    (Ca++, K+, Cl-, Protein- all counted the same)

  • Issue of pressure and cell volume regulation (cell will implode or explode otherwise)

  • The osmotic pressure is given by the equation

    P = MRT

    where P is the osmotic pressure, M is the concentration in molarity, R is the gas constant and T is the temperature


Ionic concentration

Ionic Concentration

  • Balance of Chargeand particularions

    (Ca++ counted 2x K+)

  • Maintain Electrochemical Gradient

    (negative resting potential in the cell)

  • The ionic gradient is characterized by the

    Nernst equation: DE = 58 log (C1/C2)


Osmoregulation water and solute balance

Extracellular Fluids

Electrochemical

Chemical Gradient

Negative

Charge Inside

Na+

K+

Organic

Anions

K+

Mg++

Mg++

Cl-

Ca++

Cl-

Ca++

Na+

HCO3-


Osmoregulation water and solute balance

OUTLINE:

(1) Background: Marine vs Freshwater vs Terrestrial Habitats

(2) Osmotic Pressure vs Ionic Concentration

(3) How Ionic Gradients and Osmotic constancy are maintained

(4) Ion Uptake Mechanisms


Why do osmotic and ionic concentrations have to be regulated independently

Why do osmotic and ionic concentrations have to be regulated independently?

Osmotic Concentration in and out of the cell must be fairly close

  • Animal cells are not rigid and will explode or implode with an osmotic gradient

  • Must maintain a fairly constant cell volume

    But, Ionic Concentration in and out of the cell has to be DIFFERENT:

  • Neuronal function, cell function, energy production

  • Need a specific ionic concentration in cell to allow protein functioning (protein folding would get disrupted)


How do you maintain ionic gradient but osmotic constancy

How do you maintain ionic gradient but osmotic constancy?


How do you maintain osmotic constancy but ionic difference

How do you maintain osmotic constancy but ionic difference?

A. Constant osmotic pressure:

‘Solute gap’ (difference between intra- and extracellular environments in osmotic concentrations) is filled by organic solutes, or osmolytes:

B. Difference in Ionic concentration:

(1) Donnan Effect: Use negatively charged osmolytes make cations move into cell (use osmolytes in a different way from above)

(2) Ion Transport (active and passive)


A osmotic constancy

A. Osmotic Constancy

Examples of Osmolytes:

  • Carbohydrates, such as trehalose, sucrose,

    and polyhydric alcohols, such as glycerol and mannitol

  • Free amino acids and their derivatives, including glycine, proline, taurine, and beta-alanine

  • Urea and methyl amines (such as trimethyl amine oxide, TMAO, and betaine)


B ionic gradient electrochemical gradient

B. Ionic gradient: Electrochemical Gradient

  • Donnan Effect -- use charged Osmolyte (small effect)

  • Diffusion potential -- differential permeability of ion channels (passive)

  • Active ion transport (electrogenic pumps)


Donnan effect

Donnan Effect

Osmolytes can’t diffuse across the membrane, but ions can

=

=


Donnan effect1

Donnan Effect

The negatively charged osmolyte induces cations to enter the cell and anions to leave the cell

A-

=

=

But Donnan Effect cannot account for the negative potential in the cell or for the particular ion concentrations we observe


Osmoregulation water and solute balance

Extracellular Fluids

Electrochemical

Chemical Gradient

Negative

Charge Inside

Na+

K+

Organic

Anions

K+

Mg++

Mg++

Cl-

Ca++

Cl-

Ca++

Na+

HCO3-


Osmoregulation water and solute balance

OUTLINE:

(1) Background: Marine vs Freshwater vs Terrestrial Habitats

(2) Osmotic Pressure vs Ionic Concentration

(3) How Ionic Gradients and Osmotic constancy are maintained

(4) Ion Uptake Mechanisms


Ion uptake

Ion Uptake

  • All cells need to transport ions

  • But some cells are specialized to take up ions for the whole animal

  • These cells are distributed in special organs

  • Skin, gills, kidney, gut, etc...


Ion transport

Ion Transport

  • Ion Channels

  • Facilitated Diffusion (uniport)

  • Active Transport--sets up gradient


Active transport

Active Transport

Primary Active Transport

  • Enzyme catalyses movement of solute against (uphill) an electrochemical gradient (lo->hi conc)

  • Use ATP

    Secondary Active Transport

    Symporters, Antiporters

  • One of the solutes moving downhill along an electrochemical gradient (hi-> lo)

  • Another solute moves in same or opposite directions


Primary active transport

Primary Active Transport

  • Transports ions against electrochemical gradient using “ion-motive ATPases” membrane bound proteins (enzyme) that catalyses the splitting of ATP (ATPase)

  • The enzymes form Multigene superfamilies resulting from many incidences of gene duplications over evolutionary time

Eukaryotes, Eubacteria, Archaea

Archaea

P-class ATPases are most recent while ABC ATPases are most ancient

Evolved later


Ion motive atpases

Ion-motive ATPases

  • Ion motive ATPases are present in all cells and in all taxa (all domains of life)

  • They are essential for maintaining cell function; i.e., neuronal signaling, ion-transport, energy production (making ATP), etc.


Enzyme evolution

Enzyme Evolution

  • Last time we talked about enzyme evolution in the context of evolution of function (Km and kcat) in response to temperature

  • Today, we will discuss evolution of enzyme evolution in the context of osmotic and ionic regulation (ion transport)


P class ion pumps

P-class ion pumps

P-class pumps, a gene family (arose through gene duplications) with sequence homology:

  • Na+,K+-ATPase, the Na+ pump of plasma membranes, transports Na+ out of the cell in exchange for K+ entering the cell.

  • (H+, K+)-ATPase, involved in acid secretion in the stomach, transports H+ out of the cell (toward the stomach lumen) in exchange for K+ entering the cell.

  • Ca++-ATPase, in endoplasmic reticulum (ER) & plasma membranes, transports Ca++ away from the cytosol, into the ER or out of the cell. Ca++-ATPase pumps keep cytosolic Ca++ low, allowing Ca++ to serve as a signal.

    More Info: OKAMURA, H. et al. 2003. P-Type ATPase Superfamily. Annals of the New York Academy of Sciences. 986:219-223.


Osmoregulation water and solute balance

Na+, K+-ATPase

Among the most studied of the P-class pumps is Na,K-ATPase

Professor Jens Skou published the discovery of the Na+,K+-ATPase in 1957 and received the Nobel Prize in Chemistry in 1997.


Na k atpase

Na+, K+-ATPase

  • Ion uptake, ion excretion, sets resting potential

  • Dominant in animal cells, ~25% of total energy budget

  • In gills, kidney, gut, rectal, salt glands, etc.

  • Often rate-limiting step in ion uptake

  • 3 Na+ out, 2 K+ in


Osmoregulation water and solute balance

  • Depending on cell type, there are between 800,000 and 30 million pumps on the surface of cells.

  • Abnormalities in the number or function of Na,K-ATPases are thought to be involved in several pathologic states, particularly heart disease and hypertension.


Osmoregulation water and solute balance

Axelsen & Palmgren, 1998. Evolution of substrate specificities in the P-type ATPase superfamily. Journal of Molecular Evolution. 46:84-101.

Phylogeny of P-Type ATPases

Heavy Metal

Human sequences

Black branches: bacteria, archaea

Grey branches: eukarya


Osmoregulation water and solute balance

The P-type ATPases group according to function (substrate specificity) rather than taxa (species, kingdoms)

The duplications and evolution of new function occurred prior to divergence of taxa

Possibly a few billion years ago


Osmoregulation water and solute balance

The suite of ion uptake enzymes in the gill epithelial tissue in a crab

Towle and Weihrauch, 2001


How does ion uptake activity evolve and of any of the other ion uptake enzymes

How does ion uptake activity evolve? (and of any of the other ion uptake enzymes)

  • Specific activity of the Enzyme (structural) –the enzyme itself changes in activity

  • Gene Expression and Protein synthesis (regulatory--probably evolves the fastest) –the amount of the enzyme changes

  • Localization on the Basolateral Membrane – where (which tissue or organ) is the enzyme expressed?


Osmoregulation water and solute balance

Freshwater

Stingray

Depending on the environment,

we see changes in the amount and localization of two ion uptake enzymes

V-H+-ATPaseNa+,K+-ATPase

Seawater

-acclimated

Saltwater

Stingray

Piermarini and Evans, 2001


Osmoregulation water and solute balance

Example of ion uptake Evolution

Eurytemora affinis


Osmoregulation water and solute balance

Recent invasions from salt to freshwater habitats (ballast water transport)


Osmoregulation water and solute balance

Problem: must maintain steep concentration gradient between body fluids and dilute water

Hemolymph Osmolality (mOsm/kg)

Eurytemora affinis

Surrounding water

EnvironmentalConcentration (mOsm/kg)

Lee, Posavi, Charmantier, In Prep.


The concept of a regulator2

The concept of a “Regulator”

  • Maintain constancy (homeostasis) in the face of environmental change

  • Could regulate in response to changes in temperature, ionic concentration, pH, oxygen concentration, etc…


Evolutionary shift in hemolymph concentration

Evolutionary Shift in Hemolymph Concentration

Hemolymph Osmolality (mOsm/kg)

Freshwater population can maintain significantly higher hemolymph concentration at low salinities

(0, 5 PSU; P < 0.001)

Fresh population

Saline population

5

15

25

PSU

0

mOsm/kg

EnvironmentalConcentration

Lee, Posavi, Charmantier, In Prep.


Hypothesis of freshwater adaptation evolution of ion transport capacity

Na+

Cl-

Integument

Hypothesis of Freshwater Adaptation:Evolution of ion transport capacity

Increase Ion uptake?

Adapted from Towle and Weihrauch (2001)


Models of ion transport in saline and freshwater habitats

Models of Ion Transport in Saline and Freshwater Habitats

  • In fresh water, V-type H+ ATPasecreates a H+ gradient on apical side to drive Na+ into cell against steep conc. gradient

  • Na+, K+-ATPase alone cannot provide the driving force for Na+ uptake because of thermodynamic constraints(Larsen et al. 1996)

  • In salt water, Na+ could simply diffuse into the cell, and the rate limiting step is Na+, K+-ATPase


Osmoregulation water and solute balance

Habitat Invasions

  • V-type H+ ATPaselocalization and activity has been hypothesized to be critical for the invasion of fresh water (to take up ions from dilute media), and the invasion of land (to regulate urine concentration)


Osmoregulation water and solute balance

What is the pattern of ion-motive ATPase evolution?

5 PSU

Larval Development

PSU

15

5

0

7

150

450

mOsm/kg

Enzyme Kinetics:

V-type ATPase, Na,K-ATPase activity


Osmoregulation water and solute balance

Enzyme activity of the saline population

Characteristic “U-shaped” pattern for ion-motive enzyme kinetics

N = 240 larvae/

treatment

Lee, Kiergaard, Gelembiuk, Eads, Posavi, In Review


Osmoregulation water and solute balance

Evolutionary Shifts in Enzyme Activity

V-type H+ ATPase:

Fresh populationhashigheractivity at 0 PSU (P < 0.001)

Na+,K+-ATPase:

Fresh populationhasloweractivity across salinities

(P < 0.001)

N = 240 larvae/

treatment

Lee, Kiergaard, Gelembiuk, Eads, Posavi, In Review


Osmoregulation water and solute balance

Dramatic Shift in V-ATPase Activity

V-type H+ ATPase:

Fresh populationhashigheractivity at 0 PSU (P < 0.001)


Osmoregulation water and solute balance

Decline in Na/K-ATPase Activity

Na+,K+-ATPase:

Fresh populationhasloweractivity across salinities

(P < 0.001)

N = 240 larvae/

treatment


Osmoregulation water and solute balance

V-type ATPase

  • Parallel evolution in ion uptake enzyme activity (shown in graph)

  • Parallel evolution in gene expression across clades

  • This parallelism suggests common underlying genetic mechanisms during independent invasions

Na,K-ATPase

Lee et al. Accepted


Ion uptake evolution

Ion Uptake Evolution

  • Results are consistent with a hypothesized mechanism of freshwater adaptation

  • In fresh water,V-type H+ ATPasecreates a H+ gradient on apical side to drive Na+ into cell against steep conc. gradient

  • In salt water, Na+ could simply diffuse into the cell, and the rate limiting step is Na+, K+-ATPase


Osmoregulation water and solute balance

Habitat Invasions

  • V-type H+ ATPase localization and activity has been hypothesized to be critical for the invasion of fresh water, and the invasion of land (to regulate urine concentration)

  • This study demonstrates evolution of V-type H+ ATPase function

  • What is remarkable here is the high speed to which these evolutionary shifts could occur (~50 years in the wild, only 12 generations in the laboratory)


Study questions

Study Questions

  • Why do cells need to maintain ionic gradients but osmotic constancy with the environment?

  • How do cells maintain ionic gradients but osmotic constancy with the environment?

  • What are ion uptake enzymes and how do they function to maintain homeostasis with respect to ionic and osmotic regulation?

  • What are ways in which ion uptake enzymes could evolve?


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