Osmoregulation: Water and Solute Balance

1. Osmoregulation: • Water and Solute Balance

3. OUTLINE: (1) Background: Marine vs Freshwater vs Terrestrial Habitats (2) Osmotic Pressurevs Ionic Concentration (3) HowIonic GradientsandOsmotic constancyare maintained (4) Ion Uptake Mechanisms

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. Habitat Invasions 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

10. 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

11. 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) 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 SO4-2 2.71 0.0112 CO3-2 0.14 0.0584

12. 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

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

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

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 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)

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

26. 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

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. 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)

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: 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 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

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

35. 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

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

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. 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

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. 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

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?