OSMOREGULATION WATER AND SOLUTE BALANCE.ppt

Water solubility and its importance during osmoregulation

1. Osmoregulation:Osmoregulation:
Water and Solute BalanceWater and Solute Balance

3. (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:

4. The concept of a “Regulator”

5. 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…

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

7. Life evolved in the Sea

8. 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)

9. Sea Fresh water Soil
Land
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

10. 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

11. • Lack of ions
• Greater fluctuations in Temperature, Ions, pH
• Life in fresh water is energetically more expensive
Fresh Water (vs Marine)
Marine Fresh Water
Na
+
10.81 0.0063
Mg
++
1.30 0.0041
Ca
++
0.41 0.0150
K
+
0.39 0.0023
Cl
-
19.44 0.0078
SO
4
-2
2.71 0.0112
CO
3
-2
0.14 0.0584
Ionic Composition (g/liter)

12. (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:

13. Challenges:

Osmotic concentration

Ionic concentration

14. 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.

15. 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

16. 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

17. 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
+

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

19. 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

20. K
+
Na
+
Cl-
HCO3-
Na
+
K
+
Mg
++
Mg
++
Ca
++
Ca
++
Cl
-
Organic
Anions
Extracellular FluidsExtracellular Fluids
The Cell

21. K
+
Na
+
Cl-
HCO3-
Na
+
K
+
Mg
++ Mg
++
Ca
++
Ca
++
Cl
-
Organic
Anions
Extracellular FluidsExtracellular Fluids
Negative
Potential Inside
Electrochemical
Chemical Gradient

22. Challenges:

Osmotic concentration

Ionic concentration

23. 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

24. 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)

25. K
+
Na
+
Cl-
HCO3-
Na
+
K
+
Mg
++ Mg
++
Ca
++
Ca
++
Cl
-
Organic
Anions
Extracellular Fluids
Negative
Charge Inside
Electrochemical
Chemical Gradient

26. (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:

27. 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)

28. How do you maintain ionic gradient
but osmotic constancy?

29. 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?

30. 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)

31. 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)

32. Donnan Effect
=
=
Osmolytes can’t diffuse across the membrane, but ions can

33. 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
-

34. K
+
Na
+
Cl-
HCO3-
Na
+
K
+
Mg
++ Mg
++
Ca
++
Ca
++
Cl
-
Organic
Anions
Extracellular Fluids
Negative
Charge Inside
Electrochemical
Chemical Gradient

35. (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:

36. 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...

37. Ion Transport

Ion Channels

Facilitated Diffusion (uniport)

Active Transport--sets up gradient

39. 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

40. 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
Archaea
Eukaryotes,
Eubacteria,
Archaea
Evolved later
P-class ATPases are
most recent while ABC
ATPases are most
ancient

41. 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.

42. 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)

43. 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.

44. 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.

45. 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

46. 
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.

47. Phylogeny of P-Type ATPases
Black branches: bacteria, archaea
Grey 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

48. 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

49. The suite of ion uptake
enzymes in the gill epithelial
tissue in a crab
Towle and Weihrauch, 2001

50. 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?

51. Freshwater
Stingray
Piermarini and Evans, 2001
Seawater
-acclimated
Saltwater
Stingray
V-HV-H
++
-ATPase-ATPase NaNa
++
,K,K
++
-ATPase-ATPase
Depending on the
environment,
we see changes in the
amount and
localization of two ion
uptake enzymes

52. Eurytemora affinis
Example of ion uptake
Evolution

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

54. 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

55. 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…

56. 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

57. Integument
Na
+
Cl
-
Increase Ion uptake?
Hypothesis of Freshwater
Adaptation: Evolution of ion transport
capacity
Adapted from Towle and Weihrauch
(2001)

58. •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

59. •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

60. 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
7150450mOsm/kg

61. 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

62. 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

63. Dramatic Shift in V-ATPase Activity
V-type H
+
ATPase:
Fresh population
has higher activity
at 0 PSU (P < 0.001)

64. Decline in Na/K-ATPase Activity
N = 240 larvae/
treatment
Na
+
,K
+
-ATPase:
Fresh population
has lower activity
across salinities
(P < 0.001)

65. • 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

66. 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

67. •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

68. 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?