Tongju Li, Frank ter Veld, Hartwig R. Nürnberger, Frank WehnerTongju Li, Frank ter Veld, Hartwig R. Nürnberger, Frank Wehner

Title

Hypertonicity-activated Cation Channels

in Primary Human HepatocytesPrimary Human Hepatocytes

Principles of Cell Volume Regulation

H2O

Shrinkage

hyperhypertonictonic

Swelling

hypohypotonictonic

RVD(regulatory

volumedecrease)

K+

Cl-RVI

(regulatoryvolume

increase)

Na+

Significance of Cell Volume Regulation

Homoiostasis: cope with the challenges of significant aniotonicity caused by high rates of substant transport or metabolism process.

Liver metabolism and gene expression: cell swelling favors the synthsis and/or inhibits the degradation of proteins, glycogen etc. Cell shrinkage induces opposite effect.

Apoptosis and proliferation

Epithelial transport: Na+ coupled transport

Na+-coupled transport across apical cell membrane of proximal renal tubules leads to accumulation of Na+ and substrate [e.g., amino acids (AA)] and thus to cell swelling, which activates basolateral K+ channels.

From Lang F: Physiol Rev. 1998 Jan;78(1):247-306

Cell Volume Regulation and Proliferation

A wide variety of mitogenic factors activate the Na+/H+ exchanger and/or Na+-K+-2Cl- cotransport which are expected to increase cell volume. The proliferation of cells can be cut down by inhibition of these transporters.

Osmotic alterations of cell volume indeed modify cell proliferation. Hypertonic shrinkage inhibits and slight osmotic cell swelling has been shown to accelerate cell proliferation.

Proliferation

Swelling

Cell volume Regulation and Apoptosis

Apoptosis

shrinkage

RVI

Cells displaying RVI are resistant against apoptosis.

Cell swelling reduce apoptosis and cell shrinkage increase apoptosis.

Apototic volume decrease (AVD) is an early prerequisite for apoptosis, and is mediated by activation of K+/Cl- channels: pharmacological block of these channels inhibits AVD as well as subsequent ultrastructural and biochemical events including cell death.

Cell volume regulation and apoptosis

Speculative model outlining the possible participation of three highly interlinked events in apoptotic volume decrease. Double lines depict inhibitory events, and dotted lines depictundefined relationships.

From Yu SP and Choi DW: PNAS 97 (2000) 9360–9362.

If......?

Proliferation(Tumourgenesis)

Apoptosis(Tumourdefense)

RVDAVD

swellingRVI

If the RVI mechanisms (as well as swelling) in tumor cells can be selectively blocked ......

Na+

H+

Na+

K+

Na+

2Cl-

K+

Cl-

Na+ K +Na+

Mechanisms of RVI in Rat HepatocytesRat Hepatocytes

Amiloride sensitiveGadolinium insensitive

Mechanisms of RVI in HepG2 CellsHepG2 Cells

Non-selective cation channel

Na+

H+

Na+

K+

Na+

2Cl-

K+

Cl-

Na+ K +Amiloride sensitiveGadolinium sensitive

Questions

In the human tumour cell-line HepG2, a non-selective cation channel is expressed that is the main mechanism of RVI.

Is the same channel expressed in (primary cultures of) non-tumorous human hepatocytes?

Or is the channel the same but differentially regulated in these cells?

Preparation of Primary Human HepatocytesPrimary Human Hepatocytes

Perfusion with collagenase

cell suspensionfiltration

cell debris

intact cells

dead cells

centrifugation

cryo-preservation

Preparation of Primary Human HepatocytesPrimary Human Hepatocytes

Cell suspension

25% Percoll

50% PercollPatch clamp

Patch clamp: Solutions and Protocol

V

I

Cl-

Na+Na+

Cl- K+

K+

solutions (mM)

bath pipette

NaCl 147 19

KCl 3 43.5

KGluc 28.5

CaCl2 1 0.5

MgCl2 1 3

EGTA 1

NaHepes 5 10

Hepes 5

Na2ATP 1

bath pipette E rev

(mM) (mM) (mV)

Na +152 31 40

Cl -154 69.5 -20

K +3 72 -80

Vh

(m

V)

0 2 4 6 8 10Time (s)

-100

-80

-60

-40

-20

0

20

40

60

Hypertonicity Increases Membrane Conductance

Typical recording showing the reversal activation of whole-cell membrane current by hypertonicity

Changes of membrane conductance and reversal potentials

Data are shown as mean±S.E., n =10. *: p<0.05, **:p<0.01 compared with iso

Hypertonicity Activitates Channels

-300

-200

-100

0

100

200

300

400

-80 -30 20

Vp (mV)

Ip (pA)hyper

iso

-45

-40

-35

-30

-25

-20

-15

-10

-5

0

Re

ve

rsa

l po

ten

tia

l (m

V)

**

0

1

2

3

4

5

iso hyper

Co

nd

uc

tan

ce

(n

S)

**

Effect of Amiloride (10-4 M)

Typical recording showing the effect of 100 µM amiloride on hypertonicity-induced current

The Channels are Amiloride-sensitive

-400

-300

-200

-100

0

100

200

300

400

500

-80 -30 20

Vp (mV)

Ip (p

A)

hyper

hyper + amiloride

iso*

*

*

*

*

0

1

2

3

4

5

6

Iso hyper hyper + amiloride

Co

nd

uct

ance

(n

S)

** *

-50

-40

-30

-20

-10

0

Rev

ersa

l p

ote

nti

als

(m

V)

****

Data are shown as mean±S.E, n=7. There are significant difference between iso and hyper or hyper+amiloride except for in the region of –10

mV ~ 0 mV.

The channels are Gadolinium-sensitive

-300

-250

-200

-150

-100

-50

0

50

100

150

200

-80 -30 20

Vp (mV)

Ip (p

A) Hyper

hyper + Gd3+

iso*

*

*

*

*

*

0

1

2

3

4

Co

nd

uct

ance

(n

S)

**

-70

-60

-50

-40

-30

-20

-10

0

Rev

ersa

l p

ote

nti

al

(mV

)

****

iso hyper hyper+Gd3+

Data are shown as mean±S.E, n=5. upper: * P<0.05 compared with iso or hyper + Gd3+.

Right: * P <0.05, ** P<0.01 compared with hyper.

Summary of pharmacology

Pharmacology of hypertonicity-induced current. Each compound was used at 100 µM. #: P < 0.05; ###: P < 0.001 compared with basal current (isotonicity); ***: P<0.001 compared with maximum hypertonic activation.

Permeability to NMDG+

Typical recording showing changes in current and reversal potential in response to the Na+ substitution by NMDG+

Summary of Ion-Selectivity

Summary of ion-substitution experiments. Primary results showed the channel is unpermeable to Cl-.*: P < 0.001 compared with Na+.

Rho A and Channel Activation

C3 exoenzyme (which selecitvely inactivates Rho A) completely inhibites hypertonic curretn activation.

Summary

1. Hypertonicity increased membrane conductance of primary human hepatocytes through activation of cation channels.

2. The channel is non-selective to Na+, K+, Li+, and is also permeable to NMDG+, unpermeable to Cl-.

3. The cation channel is amiloride-sensitive; and is strongly blocked by gadolinium and flufenamate;

4. Rho A is part of the signalling machinery employed in channel activation.

Non-selective channels

Gd3+ Flu Amil PK+/PNa

+ PLi+/PNa

+ PNMDG+/PNa

+

Rat hepatocytes - - + 0.7 ? ?

Human hepatocytes + + + 1.2 1.2 0.6

HepG2 + + + 2.2 ? 0

Caco-2 + + - 1 0.5 0.5

Hela + + - 1 1 0.1