Koivusalo M, Welch C, Hayashi H et al.Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J Cell Biol 188:547-563

Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
The Journal of Cell Biology (Impact Factor: 9.83). 02/2010; 188(4):547-63. DOI: 10.1083/jcb.200908086
Source: PubMed


Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na(+)/H(+) exchange. Yet, the functional relationship between Na(+)/H(+) exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na(+)/H(+) exchange, elevating cytosolic pH and stimulating Na(+) influx. Remarkably, although inhibition of Na(+)/H(+) exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na(+) influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na(+)/H(+) exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na(+)/H(+) exchange.

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    • "Indeed, while both shLuc and shR1 cells had similar amounts of total Rac1 (Input), shR1 cells had twice as much Rac1-GTP than shLuc cells (Fig. 4E). Therefore, we used the Rac1 blocker amiloride (Koivusalo et al., 2010) to assess whether Rac1 inhibition can change the effect of reggie-1 knockdown on FAs. The number of FAs in shR1 cells (but not in shLuc cells) was reduced to near normal, when cells were treated for 1 h with amiloride (Fig. 4F, G). "
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    • "Transgenic Drosophila lines expressing the pH sensor soluble mCherry-pHluorin (Koivusalo et al., 2010) (UAS-mCherry-pHluorin) were crossed to GMR-GAL4 to express the pH biosensor specifically in the developing eye. pH measurement were determined as described (Grillo-Hill et al., 2014). "
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    Full-text · Article · Mar 2015 · eLife Sciences
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    • "At lower concentrations (0.2–0.5 μM), ES3H was likely to enter the cells through a direct, energy-free mechanism; but at higher concentrations (0.5–2 μM), its translocation was an energy-dependent process, likely endocytosis induced by a specific agonistreceptor binding. In one previous study, stimulation by EGF in A431 cells was able to induce macropinocytosis (Hamasaki et al. 2004), a special mode of endocytosis that can be inhibited by actin and Na + /H + exchange inhibitors (Koivusalo et al. 2010). The translocation of S3-HBD Fig. 5 Internalization kinetics and pathway of ES3H. a Time dependency of ES3H. "
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