Osmoregulation:Osmoregulation:Water and Solute BalanceWater and Solute Balance

(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

OUTLINE:OUTLINE:

The concept of a “Regulator”

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

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

Life evolved in the Sea

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)

Sea Fresh water SoilLand

Protista X X X

Porifera X X

Cnideria X X

Ctenophora X

Platyhelminthes X X X X

Nemertea X X X

Rotifera X X X

Gastrotricha X X

Kinorhyncha X

Nematoda X X X

Nematomorpha X X

Entoprocta X X

Annelida X X X X

Mollusca X X X X

Phoronida X

Habitat Invasions

Sea Fresh water Soil Land

Bryozoa X X

Brachiopoda X

Sipunculida X

Echiuroida X

Priapulida X

Tardigrada X X X

Onychophora X X

Arthropoda X X X X

Echinodermata X

Chaetognatha X

Pogonophora X

Hemichordata X

Chordata X X X X

Habitat Invasions

• Lack of ions• Greater fluctuations in Temperature, Ions, pH• Life in fresh water is energetically more expensive

Fresh Water (vs Marine)

Marine Fresh WaterNa+ 10.81 0.0063Mg++ 1.30 0.0041Ca++ 0.41 0.0150K+ 0.39 0.0023

Cl- 19.44 0.0078SO4

-2 2.71 0.0112CO3

-2 0.14 0.0584

Ionic Composition (g/liter)

(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

OUTLINE:OUTLINE:

Challenges:

Osmotic concentration Ionic concentration

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

Universal Solvent Polar solution in which ions (but not nonpolar

molecules) will dissolve

Used for transport (blood, etc)

Animals are 60-80% water75% of the water is intracellular20% is extracellular (5-10% vascular)All the fluids contain 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+?

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?

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

K+

Na+

Cl-

HCO3-

Na+

K+

Mg++

Mg++

Ca++

Ca++

Cl-

OrganicAnions

Extracellular FluidsExtracellular Fluids

The Cell

K+Na+

Cl-HCO3-

Na+

K+

Mg++ Mg++

Ca++Ca++

Cl-

OrganicAnions

Extracellular FluidsExtracellular Fluids

NegativePotential Inside

ElectrochemicalChemical Gradient

Challenges:

Osmotic concentration Ionic 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

Balance of Charge and particular ions(Ca++ counted 2x K+)

Maintain Electrochemical Gradient(negative resting potential in the cell)

The ionic gradient is characterized by the

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

K+

Na+

Cl-HCO3-

Na+

K+

Mg++ Mg++

Ca++Ca++

Cl-

OrganicAnions

Extracellular Fluids

NegativeCharge Inside

ElectrochemicalChemical Gradient

(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

OUTLINE:OUTLINE:

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?

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)

How do you maintain osmotic constancy but ionic difference?

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: 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

=

=

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

Donnan Effect

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

=

=

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

A-

K+

Na+

Cl-HCO3-

Na+

K+

Mg++ Mg++

Ca++Ca++

Cl-

OrganicAnions

Extracellular Fluids

NegativeCharge Inside

ElectrochemicalChemical Gradient

(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

OUTLINE:OUTLINE:

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 Channels

Facilitated Diffusion (uniport)

Active Transport--sets up gradient

Active TransportPrimary 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 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

ArchaeaEukaryotes, Eubacteria,

Archaea

Evolved later

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

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

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 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.

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.

NaNa++, K, K++-ATPase-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

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.

Phylogeny of P-Type ATPases

Black branches: bacteria, archaeaGrey branches: eukarya

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

Heavy Metal

Human sequences

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 taxaPossibly a few billion years ago

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)

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?

FreshwaterStingray

Piermarini and Evans, 2001

Seawater-acclimated

SaltwaterStingray

V-HV-H++-ATPase-ATPase NaNa++,K,K++-ATPase-ATPaseDepending on the environment,

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

Eurytemora affinis

Example of ion uptake Evolution

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

Environmental Concentration (mOsm/kg)

Hemolymph Osmolality (mOsm/kg)

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

Surrounding water

Lee, Posavi, Charmantier, In Prep.

Eurytemora affinis

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

Hemolymph Osmolality (mOsm/kg)

Freshwater population can maintain significantly higher hemolymph concentration at low salinities (0, 5 PSU; P < 0.001)

Lee, Posavi, Charmantier, In Prep.Environmental Concentration

mOsm/kg

PSU5 15 250

Saline population

Fresh population

IntegumentNa+ Cl-

Increase Ion uptake?

Hypothesis of Freshwater Adaptation: Evolution of ion transport capacity

Adapted from Towle and Weihrauch (2001)

• In fresh water, V-type H+ ATPase creates 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

Models of Ion Transport in Saline and Freshwater Habitats

• V-type H+ ATPase localization 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)

Habitat Invasions

0 5 15

What is the pattern of ion-motive ATPase evolution?

Larval Development

Enzyme Kinetics: V-type ATPase, Na,K-ATPase activity

5 PSU

PSU

7 150 450 mOsm/kg

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

Enzyme activity of the saline population

N = 240 larvae/treatment

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

Evolutionary Shifts in Enzyme Activity

N = 240 larvae/treatment

V-type H+ ATPase:

Fresh population has higher activity at 0 PSU (P < 0.001)

Na+,K+-ATPase: Fresh population has lower activity across salinities (P < 0.001)

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

Dramatic Shift in V-ATPase Activity

V-type H+ ATPase:

Fresh population has higher activity at 0 PSU (P < 0.001)

Decline in Na/K-ATPase Activity

N = 240 larvae/treatment

Na+,K+-ATPase: Fresh population has lower activity across salinities (P < 0.001)

• 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

Lee et al. Accepted

Na,K-ATPase

V-type ATPase

Ion Uptake Evolution• Results are consistent with a hypothesized mechanism of

freshwater adaptation

• In fresh water, V-type H+ ATPase creates 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

• 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)

Habitat Invasions

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?