Blocking swelling‐activated chloride current inhibits mouse liver cell proliferation

Abstract

A non-transformed mouse liver cell line (AML12) was used to show that blocking swelling-activated membrane Cl- current inhibits hepatocyte proliferation. Two morphologically distinguishable cell populations exhibited distinctly different responses to hypotonic stress. Hypotonic stress (from 280 to 221 mosmol kg(-1)) to rounded, dividing cells activated an ATP-dependent, outwardly rectifying, whole-cell Cl- current, which took 10 min to reach maximum conductance. A similar anionic current was present spontaneously in 20 % of the dividing cells. Hypotonic stress to flattened, non-dividing cells activated no additional current. The Eisenman halide permeability sequence of swelling-activated anionic current in the dividing cells was SCN(-) > I(-) > Br(-) > Cl(-) > gluconate. Addition of either 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), tamoxifen or mibefradil inhibited swelling-activated anionic current. Hyperosmolarity by added sucrose inhibited the spontaneous anionic current in dividing cells. Added Cl- channel blockers NPPB (IC50 = 40 microM), DIDS (IC50 = 31 microM), tamoxifen (IC50 = 1.3 microM) and mibefradil (IC50 = 7 microM) inhibited proliferative growth of AML12 as determined by cell counts over 4 days or by protein accumulation over 2 days. Only the inhibitory effects of NPPB and mibefradil reversed with the drug washout. Hyperosmolarity by added sucrose (50 and 100 mM) also inhibited cell proliferation. Of the hydrophobic inhibitors neither NPPB at 40 microM nor tamoxifen at 1.3 microM, added for 48 h, reduced cellular ATP; however, DIDS at 31 microM significantly reduced cellular ATP with an equivalent increase in cellular ADP. We conclude that those membrane Cl- currents that can be activated by hypotonic stress are involved in mechanisms controlling liver cell growth, and that NPPB, tamoxifen and mibefradil at their IC50 for growth do not suppress the metabolism of mouse hepatocytes.

Cancer Cell Cycle Modulated by a Functional Coupling between Sigma-1 Receptors and Cl– Channels

“…Volume-regulated chloride channels underlying VRCC have been recently involved in the control of the cell cycle. For example, the inhibition of these currents by blockers such as NPPB stops cell proliferation in liver cells (43). Moreover, the expression of chloride channels is tightly regulated along the cell cycle progression in carcinoma cells (27).…”

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…[60][61][62] In particular, changes in VRAC activity seem important for the G 1 /S transition phase and many cells are halted in the G 1 phase by Cl -channel inhibitors. [63][64][65][66][67][68][69] It should be noted that conclusions about VRAC and cell cycle generally rely on data obtained with pharmacological compounds with low selectivity / specificity and should be reinvestigated now that essential components of VRAC have been cloned. 2,4 In ELA cells patch clamp experiments revealed that VRAC activity decreased as ELA cells go from G 0 to G 1 , and increased again when they go from G 1 to S 66 ( Fig.…”

Biophysical and pharmacological characterization of hypotonically activated chloride currents in cortical astrocytes

“…Shen et al, 2000;Wondergem et al, 2001). In different cell types, these currents have been given several names, including volume-activated Cl -currents (I Cl,vol ) (Nilius et al, 1994(Nilius et al, , 1996, swellingactivated Cl -currents (ICl,swell) (Ackerman et al, 1994a, b;Krapivinsky et al, 1994), or volume-regulated anion currents (VRAC) (Hubert et al, 2000;Levitan et al, 2000).…”