2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein and Melanoma

Many tumor cells are characterized by an increased net acid production. They extrude the excess protons mainly through the Na(+) /H(+) -exchanger NHE1. An increased NHE1 activity elevates the metastatic potential of tumor cells. Cell migration, a key step in the metastatic cascade, requires the formation and release of integrin-mediated cell-matrix contacts (focal adhesions). As NHE1 has been localized to focal adhesion sites, the present study tests the hypothesis that NHE1 generates measurable pH nanodomains right at focal adhesions. In order to ratiometrically measure pH close to the plasma membrane, we established a novel application of the total internal reflection fluorescence microscopy (TIRFM). Human melanoma cells were transfected with DsRed2-paxillin to identify focal adhesion sites. The pH-sensitive dyes BCECF and WGA-fluorescein were used to measure the submembranous cytosolic and the pericellular pH, respectively. Distinct pH nanodomains were found at focal adhesions, particularly at those located at the cell front, where NHE1 was concentrated. These sites featured a remarkably alkaline cytosolic and an acidic pericellular pH and thus a much steeper proton gradient across the plasma membrane compared to the rest of the cell. The generation of pH nanodomains could be assigned to NHE1-mediated H(+) export because such pH domains could not be detected in NHE1-deficient cells. Given that both integrin avidity and mechanisms contributing to adhesion turnover are pH-sensitive, we propose that pH nanodomains at focal adhesions, locally created and maintained by NHE1 activity especially at the cell front, modulate adhesion dynamics in migrating cells.

Topics: Cation Transport Proteins; Cell Line, Tumor; Cell Movement; Fluoresceins; Fluorescent Dyes; Focal Adhesions; Guanidines; Humans; Hydrogen-Ion Concentration; Luminescent Proteins; Melanoma; Microscopy, Fluorescence; Neoplasm Invasiveness; Paxillin; Recombinant Fusion Proteins; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Sulfones; Transfection; Wheat Germ Agglutinins

Hydrogen peroxide (H2O2) is considered to be a mediator of apoptotic cell death but the mechanism by which it induces apoptosis is unclear. Here, we show that cells undergoing apoptosis from exposure to H2O2 display a significant decrease in intracellular concentration of superoxide (O2-) which is associated with a reduction of the intracellular milieu, as measured by an increase in the GSH/GSSG ratio and a decrease in intracellular pH. The notion that a decrease in intracellular O2- concentration triggers apoptosis is supported by the observation that H2O2-mediated apoptosis could be retarded in cells in which the intracellular O2- concentration is maintained at or above the cellular baseline level by inhibition of the major O2- scavenger superoxide dismutase (Cu/Zn SOD). Taken together, our observations indicate that a decrease in the intracellular O2- concentration, reduction and acidification of the intracellular milieu constitute a signal for H2O2-mediated apoptosis, thereby inducing a reductive as opposed to an oxidative stress.

Topics: Apoptosis; Blotting, Western; Caspase Inhibitors; Caspases; Dose-Response Relationship, Drug; Enzyme Activation; Fluoresceins; Glutathione; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Melanoma; NADH, NADPH Oxidoreductases; Necrosis; Oxidation-Reduction; Poly(ADP-ribose) Polymerases; RNA, Antisense; Superoxide Dismutase; Superoxides; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured