Blocking swelling-activated chloride current inhibits mouse liver cell proliferation

1. A non-transformed mouse liver cell line (AML12) was used to show that bl ocking swelling activated membrane Cl- current inhibits hepatocyte prolifer ation. 2. Two morphologically distinguishable cell populations exhibited distinctl y different responses to hypotonic stress. Hypotonic stress (from 280 to 22 1 mosmol kg(-1)) to rounded, dividing cells activated an ATP-dependent, out wardly rectifying, whole-cell Cl- current, which took 10 min to reach maxim um conductance. A similar anionic current was present spontaneously in 20% of the dividing cells. Hypotonic stress to flattened, non-dividing cells ac tivated no additional current. 3. The Eisenman halide permeability sequence of swelling-activated anionic current in the dividing cells was SCN- > I- > Br- > Cl- > gluconate. 4. Addition of either 4,4'-diisothiocyanatostilloene-2,2'-disulfonate (DIDS ), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), tamoxifen or mibefr adil inhibited swelling-activated anionic current. Hyperosmolarity log adde d sucrose inhibited the spontaneous anionic current in dividing cells. 5. Added Cl- channel blockers NPPB (IC50 = 40 muM), DIDS (IC50 = 31 muM), t amoxifen (IC50 = 1.3 muM) and mibefradil (IC50 = 7 muM) inhibited prolifera tive growth of AML12 as det;er mined by cell counts over 4 days or by prote in accumulation over 2 days. Only the inhibitory effects of NPPB and mibefr adil reversed with the drug washout. Hyperosmolarity by added sucrose (50 a nd 100 mM) also inhibited cell proliferation. 6. Of the hydrophobic: inhibitors neither NPPB at 40 muM nor tamoxifen at 1 .3 muM, added for 48 h, reduced cellular ATP; however, DIDS at 31 muM signi ficantly reduced cellular ATP with an equivalent increase in cellular ADP. 7. We conclude that those membrane Cl- currents that can be activated by hy potonic stress are involved in mechanisms controlling liver cell growth, an d that NPPB, tamoxifen and mibefradil at their IC50 for growth do not suppr ess the metabolism of mouse hepatocytes.