Cellular Volume Homeostasis

1. Cellular Volume Homeostasis

2. Introduction • How cell volume is perturbed • Physiological and pathophysiological consequences of cell volume change • Cell volume regulation: recovery from swelling and shrinkage • Organic osmolyte homeostasis • Detection of cell volume changes • Detection and repair of osmotic stress-induced damage

3. www.pki.uib.no/fi/biobas/ biobas-portas/img115.jpg Cell Volume Regulation Deviations in steady-state cell volume threaten cell integrity. Cell volume regulation is a compensatory mechanism that prevents uncontrolled cell shrinkage or swelling under anisosmotic conditions.

4. 1 M NaCl 0.1 M NaCl #1 #2 H2O Piston “Semipermeable” membrane Osmotic water flow across a “semipermeable” membrane • Water flows from an area of high water activity to one of lower activity • Osmotic pressure, p, is pressure required to stop water flow • van’t Hoff relationships: p1=RT[S]1; p2=RT[S]2; Dp=RTD[S]

5. Water is in thermodynamic equilibrium across cell membranes solute pcell = pout solute solute solute • Anisosmotic volume change: induced by extracellular osmotic perturbations • Isosmotic volume change: induced by changes in cytoplasmic solute content

6. Physiology and pathophysiology of cell volume change • Physiology: all cells are exposed to isosmotic volume perturbations • Physiology: organisms and cells that live in osmotically unstable environments • Pathophysiology: e.g., systemic osmolality disturbances, anoxia and ischemia, reperfusion injury, diabetes, sickle cell crisis • intertidal zone • gut • kidney

7. Cell volume is regulated by the gain and loss of osmotically active solutes solute gain solute loss normal volume Regulatory Volume Increase (RVI) Regulatory Volume Decrease (RVD)

8. Volume regulatory electrolyte gain and loss are mediated by rapid changes in membrane transport • Advantages: allows cell to rapidly correct their volume by activating pre-existing transport pathways • Disadvantages: disruption of intracellular ion concentrations and cytoplasmic ionic strength

9. Cell volume regulation Sustained Fast Volume adjustment by salt transport across the cell membrane Volume adjustment by modification of cellular organic osmolytes (amino acids) Cell volume regulation is one prerequisite for euryhalinity!

10. Cellular volume regulation

11. Cellular volume regulation • Animal cells respond acutely to loss of water by activating Na+/H+ antiporters, Cl-/HCO3- antiporters, and/or Na+/K+/2Cl- symporter that bring potassium chloride and sodium chloride into the cell. • High internal salt concentration facilitates the entering of water and returns the cell to its original volume in minutes.

12. Long-term cellular volume control • Cells use small organic molecules called osmolytes, including amino acids, polyalcohols (sorbitol and inositol), and methylamines, to adjust the osmotic strength of cytoplasm and to maintain their volume.

13. medweb.bham.ac.uk/research/calcium/ Purpose • To study the role of calcium in regulatory volume decrease Ca2+-dependent RVD: Human ciliary epithelial cells (Adorante & Cala 1995) Choroids plexus epithelial cells (Christensen 1987) Necturus red cells (Light et al. 2003) Madin-Darby canine kidney cells (Rothstein & Mack) Ca2+-independent RVD: Rat cerebellar astrocytes (Morales-Mulia et al. 1998) Trout proximal renal tubules (Hilde & Norderhus 1998) Trout erythrocytes (Garcia-Romeu et al. 1991)

14. Is cell swelling followed by an increase in cytosolic Ca2+? Hypotonic Shock ??? Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+

15. Voltage- gated Ca2+ ? Ca2+ ? SA Investigating calcium entry • What is the mechanism by which calcium enters cells? Ca2+ ? P2 • P2 receptors may be involved in calcium entry (Light et al. 2002). • Significant amounts of calcium pass through stretch-activated channels in physiological solutions (Zou et al. 2002). • Will the stretch-activated channel antagonist inhibit volume recovery?

16. Investigating calcium entry • Does calcium entry into alligator cells occur through a P2 receptor? Ca2+ P2 • Will the ATP scavenger hexokinase and the P2 receptor blocker attenuate volume recovery?

17. Ca2+ K+ Recovered cell Swollen cell Ca2+ ? Recovered cell Swollen cell Determining calcium’s role in RVD • By what mechanism does calcium aid in volume recovery? • Is Ca2+ required to stimulate K+ efflux? • Will allowing for K+ efflux reverse the inhibitory effect of a low Ca2+ medium?

18. Examining Ca2+-mediated intracellular signaling cascades • Does calcium act as a second messenger to modulate K+ loss? Ca2+ RVD A B C Recovered cell Swollen cell • Arachidonic acid and/or its metabolites have been implicated in RVD of many cell types (Pasantes-Morales et al. 2000, Kanli & Norderhus 1998, Light et al. 1998).

19. Arachidonic acid metabolism phospholipids phospholipase A2 Ca2+ RVD arachidonic acid lipoxygenase cyclooxygenase cytochrome P450 leukotrienes EETs RVD prostaglandins RVD RVD

20. Conclusion • Calcium entry into cells occur by way of a stretch-activated channel or P2 receptor. • In cells, Ca2+ stimulates PLA2 activation and the production of arachidonic acid. • Arachidonic acid and eicosanoids, plays a role in alligator volume recovery, most likely by activating K+ efflux.

21. H2O Ca2+ cell swelling P2 PLA2 phospholipid bilayer Conclusion AA K+ Recovered cell Swollen cell

22. Amino acids Polyols Methylamines Alanine Proline Taurine Glycerol Sorbitol myo-Inositol TMAO Betaine GPC Organic osmolytes allow cells to maintain long-term stability of cytoplasmic ionic strength Three major classes of organic osmolytes:

23. Organic osmolytes are “compatible” or “non-perturbing” solutes • Compatible solutes are an ubiquitous solution to osmotic stress; used by all organisms for cellular osmoregulation • High water solubility: accumulated to cytoplasmic concentrations of 10s to 100s of millimolar • Compatible solutes do not perturb macromolecular structure or function when present at high concentrations

24. Perturbing solute Compatible solute Compatible solutes are excluded from the surface of macromolecules • No net charge at physiological pH • Lack strongly hydrophobic regions • Steric properties

25. Organic osmolyte accumulation occurs by changes in synthesis or membrane transport • Metabolically expensive: organic osmolytes are accumulated against concentration gradients of up to 107-fold

26. Organic osmolyte accumulation requires increased gene expression • Slow: requires many hours of exposure to osmotic stress

27. Organic osmolyte loss is mediated by: • Decreases in gene expression: rapid and slow components • Increase in passive efflux: rapid

28. Effector Sensor Transducer Effector How do cells detect volume changes? • Signals: mechanical stress; dilution and concentration of cytoplasmic constituents • Signal transduction: kinases and phosphatases

29. MscL channel • swelling/stretch-activated • cloned protein activated by bilayer stretch Membrane-bound enzymes ProP transporter • PLA2 • GTPases • shrinkage-activated • cloned protein activated by liposome shrinkage Model channels • alamethicin • gramicidin Mechanical stress (bilayer model): force transduction via the lipid bilayer E. coli MscL channel From Sukharev et al. Ann. Rev. Physiol. 59:633-657, 1997

30. Mechanical stress (tethered model): force transduction via cytoskeletal/extracellular proteins Chalfie, Driscoll and coworkers

31. Cytoplasmic dilution/concentration of small solutes • Intracellular ionic strength • Specific ions (e.g., K+) • Other solutes??

32. 10 8 6 Aldose reductase activity 4 2 0 100 150 200 250 300 Cell Na + K (mM) Increased cell ionic strength increases expression of organic osmolyte transporters and synthesis enzymes Uchida et al., Am. J. Physiol. 256:C614-C620, 1989

33. Isotonic Hypertonic TonEBP siRNA TonEBP siRNA Control Control Aldose reductase Na/myo-inositol cotransporter Young et al., J. Am. Soc. Nephrol. 14:283-288, 2003 The transcriptional activator TonEBP regulates hypertonicity-induced gene expression

34. Hypotonic Isotonic Hypertonic TonEBP translocates into the cell nucleus in response to hypertonic stress Lee et al., Biochem. Biophys. Res. Comm. 294:968-975, 2002

35. Regulation of gene expression by TonEBP Isotonic Hypertonic

36. Cytoplasmic dilution/concentration: macromolecular crowding and confinement • Crowding and confinement alter macromolecule thermodynamic activity, structure and interactions • Small changes in crowding and confinement can lead to large changes in the activity of signaling pathways, gene transcription, • channel/transporter activity, etc.

37. Signal transduction: the case for kinases and phosphatases Swelling-activated RVD Shrinkage-activated RVI

38. fray gene product CePASK OSR1 PASK Dan et al., Trends Cell Biol., 2001 STE20-related kinases interact with N-termini of KCl and NaK2Cl cotransporters Piechotta et al., J. Biol. Chem. 277:50812–50819, 2002

39. PASK Kinase dead PASK Rubidium influx Empty vector Time (min) PASK regulates shrinkage-induced activation of the NaK2Cl cotransporter Dowd and Forbush, J. Biol. Chem. 278:27347-27353, 2003

40. NMDG-Cl NMDG-Cl P CeGLC-7a/b CLH-3b Meiotic maturation, swelling CLH-3b Meiotic arrest, shrinkage CePASK Inactive Active 1000 pA 250 ms CePASK regulates the C. elegans volume-sensitive ClC channel CLH-3b Basal current Swelling- or meiotic maturation-induced current Denton, Rutledge, Nehrke, Strange

41. Osmosensing in yeast

42. Some consilience (finally) • Yeast STE20: shrinkage-induced activation of glycerol accumulation and RVI (turgor regulation) • CePASK: shrinkage-induced inactivation of ClC anion channel • PASK: shrinkage-induced activation of NaK2Cl cotransporter and RVI

43. How cell volume is perturbed matters Cells sense: • Extent of volume change • Rate of volume change • Mechanism of volume change (anisosmotic vs. isosmotic)

44. Volume recovery • Rapid electrolyte accumulation/loss • Organic osmolyte homeostasis • Slow accumulation • Rapid efflux/loss • Damage detection/repair • Detection • Cell cycle arrest • Repair or apoptosis The cellular osmotic stress response

46. Two kinds of water potential energy • Osmotic force: a form of chemical potential energy • Hydrostatic force: a form of mechanical potential energy • These forces are interconvertible, so the net driving force for water between a cell and the extracellular solution is RT (Osmcell - Osmext) + (Pcell – Pext)

47. Osmotic swelling is an unavoidable problem for all cells • The swelling arises from the presence of negatively-charged proteins trapped in the cytoplasm • First, imagine that a water-permeable membrane separates two rigid compartments. • One compartment has a 150 mmolal concentration of NaCl. • The other one has 150 mEq/liter of Na+ and an equal quantity of anionic charge as protein – however, the protein concentration is only 1 mmolal. • Is there an osmotic gradient? • Is there a solute gradient?

48. Gibbs-Donnan Membrane Equilibrium. • Proteins are not only large, osmotically active, particles, but they are also negatively charged anions. • Proteins influence the distribution of other ions so that electrochemical equilibrium is maintained.

50. Total Volume 100 ml 50 K+ 50 K+ Initial 50 Cl- 50 Pr - 100 Osmoles 100 Osmoles 67 K+ Ions Move 33 K+ Step 2 17 Cl- 33 Cl- 50 Pr - 66 Osmoles 134 Osmoles 67 K+ H2O moves 33 K+ Final 17 Cl- 33 Cl- 50 Pr - 33 ml 67 ml Donnan’s Law • The product of Diffusible Ions is the same on the two sides of a membrane.