Open AccessAmiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling
Author(s) -
Mirkka Koivusalo,
Christopher M. Welch,
Hisayoshi Hayashi,
Cameron C. Scott,
Moshe Kim,
Todd Alexander,
Nicolas Touret,
Klaus M. Hahn,
Sergio Grinstein
Publication year - 2010
Publication title -
the journal of cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.414
H-Index - 380
eISSN - 1540-8140
pISSN - 0021-9525
DOI - 10.1083/jcb.200908086
Subject(s) - pinocytosis , amiloride , biology , microbiology and biotechnology , endocytosis , cytosol , cdc42 , nigericin , biophysics , phosphatidylinositol , biochemistry , rac1 , staurosporine , phosphorylation , signal transduction , protein kinase c , receptor , chemistry , sodium , membrane , enzyme , organic chemistry
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.