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1. Title Tongju Li, Frank ter Veld, Hartwig R. Nürnberger, Frank Wehner Hypertonicity-activated Cation Channels in Primary Human Hepatocytes

2. Principles of Cell Volume Regulation hypotonic hypertonic Na+ K+ Shrinkage Swelling RVD (regulatory volume decrease) RVI (regulatory volume increase) Cl- H2O

3. Significance of Cell Volume Regulation • Homoiostasis: cope with the challenges of significant aniotonicity caused by high rates of substant transport or metabolism process. • 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 • 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

4. Cell Volume Regulation and Proliferation Proliferation Swelling 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 acceleratecell proliferation.

5. Cell volume Regulation and Apoptosis shrinkage RVI Apoptosis 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 thesechannels inhibits AVD as well as subsequent ultrastructural and biochemical events including cell death.

6. Cell volume regulation and apoptosis Speculative model outlining the possible participationof three highly interlinked events in apoptotic volume decrease. Double lines depict inhibitory events, and dotted lines depict undefined relationships. From Yu SP and Choi DW: PNAS 97 (2000) 9360–9362.

7. If......? Proliferation (Tumourgenesis) swelling RVI AVD RVD Apoptosis (Tumourdefense) If the RVI mechanisms (as well as swelling) in tumor cells can be selectively blocked ......

8. Mechanisms of RVI in Rat Hepatocytes Na + H + Na + Na+ K + 2Cl - Na + K + Na + Cl - K + Amiloride sensitive Gadolinium insensitive

9. Mechanisms of RVI in HepG2 Cells Na + H + Na + K + 2Cl - Na + K + Na + Cl - K + Non-selective cation channel Amiloride sensitive Gadolinium sensitive

10. 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?

11. Preparation of Primary Human Hepatocytes Perfusion with collagenase filtration cell suspension

12. Preparation of Primary Human Hepatocytes Patch clamp dead cells centrifugation Cell suspension 25% Percoll 50% Percoll intact cells cell debris cryo-preservation

13. 60 40 bath pipette E 20 Vh (mV) rev (mM) (mM) (mV) 0 + Na 152 31 40 -20 - Cl 154 69.5 -20 + K -40 3 72 -80 -60 -80 I -100 0 2 4 6 8 10 Time (s) V K+ Na+ Na+ Cl- Cl- K+ Patch clamp: Solutions and Protocol

14. Hypertonicity Increases Membrane Conductance Typical recording showing the reversal activation of whole-cell membrane current by hypertonicity

15. hyper iso Hypertonicity Activitates Channels 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

16. Effect of Amiloride (10-4 M) Typical recording showing the effect of 100 µM amiloride on hypertonicity-induced current

17. * * 6 * * 5 hyper hyper + amiloride iso * 4 * 3 Conductance (nS) * * 2 1 0 Iso hyper hyper + amiloride 0 -10 -20 Reversal potentials (mV) -30 ** ** -40 -50 The Channels are Amiloride-sensitive 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.

18. 4 3 2 * Conductance (nS) Hyper hyper + Gd3+ iso * * * 1 * * 0 iso hyper hyper+Gd3+ 0 * -10 * -20 -30 Reversal potential (mV) -40 -50 -60 ** ** -70 The channels are Gadolinium-sensitive 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.

19. 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.

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

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

22. Rho A and Channel Activation C3 exoenzyme (which selecitvely inactivates Rho A) completely inhibites hypertonic curretn activation.

23. Summary • Hypertonicity increased membrane conductance of primary human hepatocytes through activation of cation channels. • The channel is non-selective to Na+, K+, Li+, and is also permeable to NMDG+, unpermeable to Cl-. • The cation channel is amiloride-sensitive; and is strongly blocked by gadolinium and flufenamate; • Rho A is part of the signalling machinery employed in channel activation.

24. 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 Non-selective channels