Regulation of the formation of tumor cell pseudopodia by the Na+/H+ exchanger NHE1

The Na+/H+ exchanger NHE1 is involved in intracellular pH homeostasis and c ell volume regulation and accumulates with actin in the lamellipodia of fib roblasts, In order to determine the role of NHE1 following epithelial trans formation and the acquisition of motile and invasive properties, we studied NHE1 expression in polarized MDCK cells, Moloney Sarcoma virus (MSV) trans formed MDCK cells and an invasive MSV-MDCK cell variant (MSV-MDCK-INV), Exp ression of NHE1 was significantly increased in MSV-MDCK-INV cells relative to MSV-MDCK and MDCK cells. NHE1 was localized with p-actin to the tips of MSV-MDCK-INV cell pseudopodia by immunofluorescence. Sensitivity of NHE1-me diated Na-22 uptake to ethylisopropylamiloride, a specific inhibitor of NHE 1, was increased in MSV-MDCK cells relative to MDCK cells. Changes in intra cellular pH induced upon EIPA treatment were also of higher magnitude in MS V-MDCK and MSV-MDCK-INV cells compared to wild-type MDCK cells, especially in Hepes-buffered DMEM medium. Inhibition of NHE1 by 50 muM ethylisopropyla miloride induced the disassembly of actin stress fibers and redistribution of the actin cytoskeleton in all cell types. However, in MSV-MDCK-INV cells , the effect of ethylisopropylamiloride treatment was more pronounced and a ssociated with the increased reversible detachment of the cells from the su bstrate. Videomicroscopy of MSV-MDCK-INV cells revealed that within 20 minu tes of addition, ethylisopropylamiloride induced pseudopodial retraction an d inhibited cell motility, The ability of ethylisopropylamiloride to preven t nocodazole-induced formation of actin stress fibers in MSV-MDCK cells was more pronounced in Hepes medium relative to NaHCO3 medium, showing that NH E1 can regulate actin stress fiber assembly in transformed MSV-MDCK cells v ia its intracellular pH regulatory effect. These results implicate NHE1 in the regulation of the actin cytoskeleton dynamics necessary for the adhesio n and pseudopodial protrusion of motile, invasive tumor cells.