ABSTRACT
Volume-activated Cl- channels (VACCs) can be activated by hypotonic solutions and have been identified in many cell types. Here, we investigated the effects of different statins on VACCs in monocytes. Whole-cell patch clamp recordings demonstrated that a hypotonic solution induced 5-nitro-2- (3-phenylpropylamino) benzoic acid (NPPB)- and 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive VACC currents in human peripheral monocytes and RAW 264.7 cells. The VACC currents were inhibited by the lipophilic statin (simvastatin) but not by the hydrophilic simvastatin acid and pravastatin. A low-molecular-weight superoxide anion scavenger (tiron, 1 mM) and inhibitor of NADPH oxidase (DPI 10 µM) was able to abolish the VACC currents. A hypotonic solution increased the reactive oxygen species (ROS) detected by the fluorescence of dichlorodihydrofluorescein (DCF), which was abolished by tiron and DPI. NPPB, DIDS, and simvastatin but not pravastatin decreased the fluorescence of DCF. Simvastatin could not further decrease VACC currents when pretreated with tiron or DPI, whereas exogenous H2O2 (100 µM), increased the VACC currents and overcame the blockade of VACC currents by simvastatin. Functionally, hypotonic solution increased the TNF-α mRNA expression, which could be decreased by tiron, DPI, NPPB, DIDS and simvastatin but not pravastatin. However, simvastatin could not decrease the TNF-α expression further when pretreatment with tiron, DPI, NPPB or DIDS. We conclude that lipophilic (simvastatin) rather than hydrophilic statin inhibit VACCs and decrease hyposmolality induced inflammation in monocytes by inhibiting NADPH oxidase.
Subject(s)
Chloride Channels/drug effects , Chloride Channels/physiology , Monocytes/physiology , NADPH Oxidases/antagonists & inhibitors , Simvastatin/chemistry , Simvastatin/pharmacology , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Humans , Hydrophobic and Hydrophilic Interactions , Hydroxymethylglutaryl-CoA Reductase Inhibitors/chemistry , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hypotonic Solutions/chemistry , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Lipids/chemistry , Mice , Monocytes/drug effects , NADPH Oxidases/metabolism , Osmotic Pressure , RAW 264.7 CellsABSTRACT
Extracellular acidic pH activated chloride channels (I(Cl,acid)) have been characterized in HEK 293 cells and mammalian cardiac myocytes. This study was designed to evaluate the expression of I(Cl,acid) in RAW264.7 monocytic-macrophage and human peripheral monocytes and to investigate the effect of simvastatin on I(Cl,acid). In two kinds of cells, the activation and deactivation of the current rapidly and repeatedly followed the change of the extracellular solution to pH=4.3. Compared with the outward current (pA/pF) activated at pH 4.3, the currents inhibited by simvastatin at concentrations of 0.1 microM were all decreased a little, however the currents at concentrations of 1 microM and 10 microM simvastatin were decreased significantly. The IC(50) for simvastatin inhibiting I(Cl,acid) of RAW264.7 was 13.77 microM. In summary, we report for the first time that simvastatin inhibits the I(Cl,acid) of RAW264.7 monocytic-macrophage and human peripheral monocytes in a concentration-dependent manner.
