Cellular Volume Homeostasis
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Cellular Volume Homeostasis. 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

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Cellular Volume Homeostasis

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Cellular Volume Homeostasis


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


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.


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]


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


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


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)


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


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!


Cellular volume regulation


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.


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.


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)


  • Is cell swelling followed by an increase in cytosolic Ca2+?

Hypotonic Shock

???

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+


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?


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?


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?


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).


Arachidonic acid metabolism

phospholipids

phospholipase A2

Ca2+

RVD

arachidonic acid

lipoxygenase

cyclooxygenase

cytochrome P450

leukotrienes

EETs

RVD

prostaglandins

RVD

RVD


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.


H2O

Ca2+

cell swelling

P2

PLA2

phospholipid

bilayer

Conclusion

AA

K+

Recovered cell

Swollen cell


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:


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


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


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


Organic osmolyte accumulation

requires increased gene expression

  • Slow: requires many hours of exposure to osmotic stress


Organic osmolyte loss is mediated by:

  • Decreases in gene expression: rapid and slow components

  • Increase in passive efflux: rapid


Effector

Sensor

Transducer

Effector

How do cells detect volume changes?

  • Signals: mechanical stress; dilution and concentration of cytoplasmic constituents

  • Signal transduction: kinases and phosphatases


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


Mechanical stress (tethered model): force transduction via cytoskeletal/extracellular proteins

Chalfie, Driscoll and coworkers


Cytoplasmic dilution/concentration of small solutes

  • Intracellular ionic strength

  • Specific ions (e.g., K+)

  • Other solutes??


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


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


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


Regulation of gene expression by TonEBP

Isotonic

Hypertonic


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.


Signal transduction: the case for

kinases and phosphatases

Swelling-activated

RVD

Shrinkage-activated

RVI


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


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


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


Osmosensing in yeast


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


How cell volume is perturbed matters

Cells sense:

  • Extent of volume change

  • Rate of volume change

  • Mechanism of volume change (anisosmotic vs. isosmotic)


  • 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


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)


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?


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.


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.


Gibbs Donnan equilibrium

The equilibrium characterized by an unequal distribution of diffusible ions between two ionic solutions separated by a membrane which is impermeable to at least one of the ionic species present

A

A

B

B

Na+

Na+

[Na+]

[Na+]

=

1

1

Cl-

[Cl-]

[Cl-]

=

A

A

B

B

2

Na+

Na+

[Na+]

[Na+]

>

2

1

Pr-

Cl-

[Cl-]

[Cl-]

<

Pr-

1) Cl- moves to side A by concentration gradient.

2) Side A becomes negative, attracting therefore Na+ by electrical gradient

→ Unequal distribution of diffusible ions


Double Donnan equilibrium

Unequal distribution of diffusible ions by Gibbs Donnan equilibrium

→ Donnan excess

→ Increase in intracellular osmotic pressure

→ Cell lysis by water movement

3 Na+

Na+

2 K+

Na+

Pr-

K+

K+

Cl-

"Double Donnan equilibrium"

Cl-


Initial conditions

Intermediate conditions: Cl- diffused

down its gradient; why did Na+ move

against its gradient? Notice that there is now a gradient of electrical charge – this is a Donnan potential.

Now imagine water trying to move osmotically – is there a gradient of hydrostatic pressure? The system has come into Gibbs-Donnan equilibrium – all forces are balanced.


Animal cells could never attain Gibbs-Donnan Equilibrium

  • Why not? The plasma membrane cannot sustain a hydrostatic pressure gradient.

  • Without the evolution of some means of avoiding Gibbs-Donnan equilibrium, there would be no protein-containing cells.


ELECTROCHEMICAL GRADIENT AND EQUILIBRIUM

A

B

0.1 M K+

0.01 M K+

0.1 M Cl-

0.01 M Cl-


GIBBS-DONNAN EQUILIBRIUM

A

B

0.1 M K+

0.1 M K+

0.1 M Cl-

0.1 M Anions


The Na+/K+ Pump counteracts G-D equilibration

The Na+/K+ pump undergoes cycles in which it spends an ATP to eject 3 Na+ from the cell and at the same time to take 2 K+ into the cell. On the average, this counteracts leakage of Na+ and K+ across the membrane down their electrochemical gradients. The bottom-line effect of this is to make the cell effectively impermeable to NaCl. Gibbs-Donnan equilibrium is not approached and the cell does not swell, in spite of the presence of protein anion (X-).


What if the Na+/K+ pump stops working?


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