Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling.
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.
Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Mirkka
M
Koivusalo
Christopher
C
Welch
Hisayoshi
H
Hayashi
Cameron C
CC
Scott
Moshe
M
Kim
Todd
T
Alexander
Nicolas
N
Touret
Klaus M
KM
Hahn
Sergio
S
Grinstein
D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D051380:GRB2 Adaptor Protein / Q000378:metabolism; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010873:Pinocytosis / Q000187:drug effects*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors; D013450:Sulfones / Q000494:pharmacology; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D051339:Actin Depolymerizing Factors; D051380:GRB2 Adaptor Protein; D006146:Guanidines; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; C074107:3-methylsulfonyl-4-piperidinobenzoyl guanidine; D004815:Epidermal Growth Factor; D000584:Amiloride; D012964:Sodium; D051057:Proto-Oncogene Proteins c-akt; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein